Substituted stilbenes and their reactions

ABSTRACT

The present invention relates to stilbene and quinine compounds related to combretastatin A-4 and their use as anticancer compounds and prodrugs. The compounds include those with an alkyl group on the double bond of cis- or trans-stilbenes, compounds with one or more (and preferably 2 or 3) alkyl group substituents on the stilbene A ring, compounds with an alkoxy group other than methoxy at position 3, 4, and/or 5 of the stilbene A ring, compounds (or prodrugs) in which BBOC amino acid esters are formed with the phenolic hydroxyl at the 3-position of the B ring and compounds (or prodrugs) based on a benzoquinone B ring. The present invention further relates to the photochemical reactions of stilbene compounds, either the above compounds disclosed for the first time herein or compounds based on prior stilbenes. These reactions include the photochemical release of an active form of the compound from a prodrug conjugate and the photochemical isomerization of the compounds, especially from a trans to cis form of compounds. The reactions can be used alone or in combination to convert inactive or comparatively less active forms of the compounds to more active forms, thereby allowing the compounds to be selectively targeted, e.g., activating them at the site of a tumor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/744,405, filed May 4, 2007, which is a continuation of U.S. patentapplication Ser. No. 10/451,213, filed Mar. 3, 2004 (now U.S. Pat. No.7,220,784, issued May 22, 2007), which is the U.S. National Stage ofInternational Application No. PCT/GB01/05702, filed Dec. 20, 2001. Thedisclosures of the aforesaid applications are incorporated by referencein their entireties in the present application.

FIELD OF THE INVENTION

The present invention relates to novel compounds, and more particularlyto stilbene and quinone compounds related to combretastatin A-4 andtheir possible use as anticancer compounds and prodrugs. In furtheraspects, the present invention relates to the photochemical reactions ofsome of these compounds, in the photochemical isomerisation of thecompounds and/or the photochemical release of an active compound from aprotected compound (prodrug).

BACKGROUND OF THE INVENTION

The stilbene cis-combretastatin A-4 Z-1, isolated from the African bushwillow, Combretum caffrum shows exciting potential as an anticanceragent, binding strongly to tubulin and displaying potent and selectivetoxicity toward tumour vasculature (U.S. Pat. No. 4,996,237, ArizonaBoard of Regents, Pettit et al, Experimentia, 1989, 45, 209; Lin et al,Mol. Pharmacol., 1988, 34, 200; Grosios et al, Brit. J. Cancer, 1999,81, 1318; Lin et al, Biochemistry, 1989, 28, 6984; Woods et al, Brit. J.Cancer, 1995, 71, 705; McGown et al, Cancer Chemother. Pharmacol., 1990,26, 79; El-Zayat et al, Anti-Cancer Drugs, 1993, 4, 19; Dark et al,Cancer Research, 1997, 57, 1829.).

Cis-combretastatin A-4 Z-1 is able to inhibit cell growth at lowconcentrations (IC₅₀, P388 murine leukaemia cell line 2.6 nM). Thepotency of trans-combretastatin A-4 E-1 is much lower and inhibits cellgrowth in the μM range. Arguably, it is the ability of Z-1 and Z-2 todestroy tumour blood vessels, effectively starving tumours of nutrients,which makes them such exciting molecules. Tumour vasculature and theformation of neovasculature were first identified as a target for cancertherapy by Judah Folkman some 30 years ago. The work of Folkman andothers has clearly identified angiogenesis and blood supply as necessaryrequirements for primary tumour growth, invasiveness and metastasis. Itis now becoming clear that the selective destruction of tumourvasculature will have a significant impact on the clinical treatment ofcancer. Angiogenesis is subject to a complex process of regulation andthereby offers a multitude of molecular targets for drug design.

The use of Z-1 as a clinically useful anticancer agent has been severelyhampered by its poor water solubility (Brown et al, J. Chem. Soc.,Perkin Trans. 1, 1995, 577). The phosphate salt Z-2 is more soluble inwater than Z-1 and is soon to enter phase II clinical trials (Pettit etal, Anti-Cancer Drug Des., 1995, 10, 299). Nevertheless, both Z-1 andZ-2 are not targeted towards cancer cells and their therapeutic efficacywould be improved if their selectivity were better. The low solubilityof cis-combretastatin A-4 in water and saline has led to attempts in theart to make related compounds or prodrugs which retain the activity ofcis-combretastatin A-4 as an anticancer agent and which have enhancedsolubility. These attempts focus on forming salts or derivatives at thephenolic hydroxyl group of combretastatin. By way of example, U.S. Pat.No. 5,561,122 (Arizona Board of Regents) discloses the sodium andpotassium salts of cis-combretastatin A-4 and a hemisuccinic acid esterderivative, and WO99/35150 (Arizona Board of Regents) discloses thelithium, caesium, magnesium, calcium, manganese and zinc salts ofcis-combretastatin A-4, and ammonium cation salts with imidazole,morpholine, piperazine, piperidine, pyrazole, pyridine, adenosine,cinchonine, glucosamine, quinine, quinidine, tetracycline and verapamil.

At the molecular level, both compounds target tubulin, binding stronglyat or close to the colchicine (3) binding site, preventingpolymerisation of α,β-tubulin heterodimer to microtubules. Theirinhibition of microtubule formation prevents mitosis and is important indisrupting the growth of new vascular epithelial cells. In addition,disruption of the intracellular microtubule networks by combretastatinA4 leads to the destruction of microvessels within the tumour. Thisantivascular activity offers exciting therapeutic possibilities as thedestruction of microvessels results in the death of all tumour cellswhich depend on the vessel for nutrients and oxygen. Themulti-functional role of tubulin in both healthy and cancer cellshighlights the need for selectively targeted drugs.

We have previously investigated the tubulin-binding properties of agentsrelated to Z-1 and 3 and as part of this effort, we have designed manyrelated compounds that behave in a similar fashion to Z-1 (Ducki et al,Bioorg. Med. Chem. Lett., 1998, 8, 1051; Zhao et al, Eur. J. Nuc.Medicine, 1999, 26, 231; Aleksandrzak et al, Anti-Cancer Drugs, 1998, 9,545). However, it remains a problem in the art in designing effectivecompounds and especially those which can be selectively targeted.

SUMMARY OF THE INVENTION

In a first group of aspects, the present invention relates to novelcompounds and more particularly to stilbene and quinone compoundsrelated to combretastatin A-4. The synthesis of new compounds isdisclosed herein, together with experiments demonstrating their activityin vitro and in vivo, supporting their use as anticancer compounds andprodrugs. The compounds include those with an alkyl group on the doublebond of cis or trans-stilbenes, compounds with one or more (andpreferably 2 or 3) alkyl group substituents on the stilbene A ring,compounds with an alkoxy group other than methoxy at position 3, 4,and/or 5 of the stilbene A ring, compounds (or prodrugs) in which BOCamino acid esters are formed with the phenolic hydroxyl at the3-position of the B ring and compounds (or prodrugs) based on abenzoquinone B ring.

In a further group of aspects, the present invention relates to thephotochemical reactions of stilbene compounds, either the abovecompounds disclosed for the first time herein or compounds based onprior art stilbenes. These reactions include the photochemical releaseof an active form of the compound from a prodrug conjugate and thephotochemical isomerisation of the compounds, especially from a trans tocis form of compounds. The reactions can be used alone or in combinationto convert inactive or comparatively less active forms of the compoundsto more active forms, thereby allowing the compounds to be selectivelytargeted, e.g. activating them at the site of a tumour.

Accordingly, in a first aspect, the present invention provides acompound represented by the structural formula:

wherein:

-   -   X is selected from hydroxyl, nitro, amino, aryl, heteroaryl,        alkyl, alkoxy, CHO, COR, halogen, haloalkyl, NH₂, NHR, NRR′, SR,        CONH₂, CONHR, CONHRR′, O-aryl, O-heteroaryl or O-ester;    -   R₁ is selected from alkyl, CHO, alkoxy, NH₂, NHR, NRR′, SR, CF₃        or halogen;    -   R₂ and R₃ are independently selected from hydrogen, alkyl,        alkoxy, hydroxyl NH₂, NHR, NRR′, SR, haloalkyl or halogen;    -   R₄ and R₅ are independently selected from hydrogen, alkyl,        CH₂NHCOR″ or CH₂CONHR″; and,    -   R₆, R₇ and R₈ are independently selected from hydrogen, alkyl or        alkoxy;    -   or a salt or derivative thereof.

In all aspect of the invention, preferably, the substituents are chosenaccording to the following lists of preferred groups.

Preferably, alkyl or alkoxy substituents are substituted orunsubstituted C₁₋₁₀ alkyl or alkoxy groups. In either case, the alkylchain can be straight chain or branched.

Preferred alkyl substituents are methyl or ethyl. Preferred alkoxysubstituents are methoxy or ethoxy.

Halogen substituents can be fluorine, chlorine, bromine or iodine, andare preferably fluorine. Preferably, the haloalkyl groups arefluoroalkyl, and most preferably is a CF₃ group.

Preferably, the O-ester group is represented by the formula O-phosphate,OCO-alkyl, OCO-aryl, OCO-heteroaryl, OCO-amino acid, OCO-peptide,OCO-polymer, OCO-sugar or OCO—CHR—NH—BOC, where BOC represents at-butoxycarbonyl group.

As used herein, preferably R and R′ are substituted or unsubstitutedC₁₋₁₀ alkyl groups. R″ is preferably selected from substituted orunsubstituted alkyl (e.g. C₁₋₁₀), aryl or heteroaryl groups.

In a further aspect, the present invention provides compounds in whichthere are one or more alkyl groups present on the double bond linkingthe stilbene A and B rings. Thus, in this aspect, the present inventionprovides compounds represented by the structural formula:

wherein:

-   -   the zigzag line indicates that the compound can be cis or trans;    -   X is selected from hydroxyl, nitro, amino, aryl, heteroaryl,        alkyl, alkoxy, CHO, COR, halogen, haloalkyl, NH₂, NHR, NRR′, SR,        CONH₂, CONHR, CONHRR′, O-aryl, O-heteroaryl or O-ester;    -   R₁ is selected from alkyl, CHO, alkoxy, NH₂, NHR, NRR′, S, CF₃        or halogen;    -   R₂ and R₃ are independently selected from hydrogen, alkyl,        alkoxy, hydroxyl, NH₂, NHR, NRR′, SR, haloalkyl or halogen;    -   R₄ and R₅ are independently selected from hydrogen, alkyl,        CH₂NHCOR″ or CH₂CONHR″; and,    -   R₆, R₇ and R₈ are independently selected from hydrogen, alkyl or        alkoxy;        wherein at least one of the substituents R₄ and R₅ is an alkyl        group.    -   or a salt or derivative thereof.

As defined above, the compounds in this aspect of the invention may beeither the cis or Z-isomer, i.e. be related to combretastatin A4, or thetrans or E-isomer. Examples of the synthesis of both isomers are provedbelow. Preferably, the alkyl group R₄ and/or R₅ is a methyl or ethylgroup.

In a further aspect, the present invention provides compounds in whichone or more of the methoxy groups on the A ring of combretastatin isreplaced by an alkyl group. Thus, in this aspect, the present inventionprovides compounds represented by the structural formula:

wherein:

-   -   X is selected from hydroxyl, nitro, amino, aryl, heteroaryl,        alkyl, alkoxy, CHO, COR, halogen, haloalkyl, NH₂, NHR, NRR′, SR,        CONH₂, CONHR, CONHRR′, O-aryl, O-heteroaryl or O-ester;    -   R₁ is selected from alkyl, CHO, alkoxy, NH₂, NHR, NRR′, SR, CF₃        or halogen;    -   R₂ and R₃ are independently selected from hydrogen, alkyl,        alkoxy, hydroxyl, NH₂, NHR, NRR′, SR, haloalkyl or halogen;    -   R₄ and R₅ are independently selected from hydrogen, alkyl,        CH₂NHCOR″ or CH₂CONHR″; and,    -   wherein R₆, R₇ and R₈ are independently selected from hydrogen,        alkyl or alkoxy such that at least one of these substituents is        an alkyl group;    -   or a salt or derivative thereof.

In preferred embodiment, two or more preferably all three of the groupsare alkyl groups. Exemplary compounds include those with methyl, ethylor propyl groups. In a preferred embodiment, R₆, R₇ and R₈ are methylgroups.

In a further aspect, the present invention provides compounds in whichone or more of the methoxy groups on the A ring of combretastatin isreplaced by a higher alkoxy group, i.e. an ethoxy or longer chain group.Thus, in this aspect, the present invention provides compoundsrepresented by the structural formula:

wherein:

-   -   X is selected from hydroxyl, nitro, amino, aryl, heteroaryl,        alkyl, alkoxy, CHO, COR, halogen, haloalkyl, NH₂, NHR, NRR′, SR,        CONH₂, CONHR, CONHRR′, O-aryl, O-heteroaryl, or O-ester;    -   R₁ is selected from alkyl, CHO, alkoxy, NH₂, NHR, NRR′, SR, CF₃        or halogen;    -   R₂ and R₃ are independently selected from hydrogen, alkyl,        alkoxy, hydroxyl, NH₂, NHR, NRR′, SR, haloalkyl or halogen;    -   R₄ and R₅ are independently selected from hydrogen, alkyl,        CH₂NHCOR″ or CH₂CONHR″; and,    -   wherein R₆, R₇ and R₈ are independently selected from hydrogen,        alkyl or alkoxy such that at least one of these substituents is        an alkoxy group other than methoxy group;    -   or a salt or derivative thereof.

Preferably, two or all three of the groups is replaced by an alkoxygroup other than methoxy.

In a further aspect, the present invention relates to compounds in whichthe phenolic hydroxyl group on the B ring of the combretastatin isderivatised to form a t-BOC-amino acid ester. These compounds may beprodrugs capable of releasing combretastatin, or a variant thereof, e.gby the action of an enzyme capable of hydrolysing the BOC-amino acidester, e.g. an esterase enzyme. Thus, in this aspect, the presentinvention provides compounds represented by the structural formula:

wherein:

-   -   R₁ is selected from alkyl, alkoxy, NH₂, NHR, NRR′, SR, CF₃, CHO        or halogen;    -   R₂ and R₃ are independently selected from hydrogen, alkyl,        alkoxy, hydroxyl, NH₂, NHR, NRR′, SR, haloalkyl or halogen;    -   R₄ and R₅ are independently selected from hydrogen, alkyl,        CH₂NHCOR″ or CH₂CONHR″; and,    -   R₆, R₇ and R₈ are independently selected from hydrogen, alkyl or        alkoxy; and,    -   or a salt or derivative thereof;    -   wherein X is a group represented by:

wherein BOC represents a t-butoxycarbonyl group and the A group is anamino acid side chain.

The BOC amino acid ester may include a naturally occurring or syntheticamino acid, in either the D or L-isoform. Examples of compounds of theaspect of the invention include those where the amino acid is Phe, Ile,Gly, Trp, Met, Leu, Ala, His, Pro, D-Met, D-Trp, or Tyr, e.g. when incompound 33 the amino acid is Phe, the A group is —CH₂Ph etc.

In a further aspect, the present invention provides compounds in whichthe B ring of combretastatin is replaced by a substituted orunsubstituted benzoquinone ring. These quinone compounds may act asprodrugs of combretastatin and be activated in vivo by enzymes such asDT-diaphorase. Thus, in this aspect, the present invention providescompounds represented by the structural formula:

wherein:

-   -   the dotted line indicates a single or double covalent bond and        the zigzag line indicates that the compound can be cis or trans;    -   R₁, R₂ and R₃ are independently selected from hydrogen, alkyl,        CHO, COR, alkoxy, hydroxyl, NH₂, NHR, NRR′, SR, haloalkyl or        halogen;    -   R₄ and R₅ are independently selected from hydrogen, alkyl,        CH₂NHCOR″ or CH₂CONHR″; and,    -   R₆, R₇ and R₈ are independently selected from hydrogen, alkyl or        alkoxy; or    -   a salt or derivative thereof.

The present invention also includes compositions comprising one or moreof the above defined compounds. In other aspects, the present inventionprovides the compounds for use in a method of medical treatment and theuse of the compounds for the preparation of a medicament for thetreatment of a condition that responds to the medicament, and inparticular for the treatment of cancer. The compounds may act directlyor be prodrugs capable of releasing an active form of the compound uponhydrolysis or reduction, e.g. as mediated in situ by an enzyme.

In the second group of aspects, the present invention provides a prodrugcomprising a compound conjugated to a photocleavable group, wherein theprodrug is represented by the general formula:

wherein:

-   -   R₁ is selected from alkyl, CHO, alkoxy, NH₂, NHR, NRR′, SR, CF₃        or halogen;    -   R₂ and R₃ are independently selected from hydrogen, alkyl,        alkoxy, hydroxyl, NH₂, NHR, NRR′, SR, haloalkyl or halogen;    -   R₄ and R₅ are independently selected from hydrogen, alkyl,        CH₂NHCOR″ or CH₂CONHR″; and,    -   R₆, R₇ and R₈ are independently selected from hydrogen, alkyl or        alkyoxy;    -   Y is selected from O, S, Se, NH; and,    -   Z is a photocleavable group;    -   or a salt or derivative thereof.

The compounds conjugated to the photocleavable group to form the prodrugmay be the new combretastatin derivatives disclosed herein or may be aknown combretastatin which has not be conjugated in this way in theprior art.

The prodrugs can be activated by exposure to electromagnetic radiation,especially ultraviolet-visible light (e.g. having a wavelength ofbetween about 190-1000 nm), to remove the protecting photocleavablegroup and cause the release of the compound. Thus, the prodrugs can beused to provide selective activation of the active form of the compound,e.g. at the site of a tumour, by administering the compound and exposingto light the site at which activation is required.

Particularly preferred compounds (prodrugs) are those which can beexposed to light to release combretastatin, and especiallycis-combretastatin A4.

Examples of preferred compounds include those which Z, thephotocleavable group is selected from:

In the above formulae, R is the photoprotected group, R₉ and R₁₀ areindependently selected from alkyl, aryl or heteroaryl and X is anyfunctional group. Examples of photoactivatable groups are also providedon pages 54-59.

In a further aspect, the present invention provides the compounds asdefined herein for use in a method of medical treatment. In preferredembodiments, the present invention provides the use of the compoundsdefined herein for the preparation of a medicament for the treatment ofa condition that is ameliorated by administration of the activated orreleased form of the compound. In such uses, it is preferred that theactivated form of the compound has significantly greater activity thanthe protected form of the compound, e.g. making it possible to obtainselectivity in the delivery and activation of the compound, e.g. to atarget tissue. In preferred embodiments of the invention, the compoundsare employed in medicaments for the treatment of cancer.

In a further aspect, the present invention provides a process forproviding the compound at a site, the process comprising exposing aprodrug represented by the above formula to light to release thecompound at the site. In this embodiment of the invention, preferablythe light is in the visible range, e.g. from about 350-800 nm.

In a further aspect, the present invention provides a process forisomerising a compound represented by the general formula:

wherein:

-   -   R₁ is selected from alkyl, alkoxy, CHO, NH₂, NHR, NRR′, SR, CF₃        or halogen;    -   R₂ and R₃ are independently selected from hydrogen, alkyl,        alkoxy, hydroxyl, NH₂, NHR, NRR′, SR, haloalkyl or halogen;    -   R₄ and R₅ are independently selected from hydrogen, alkyl,        CH₂NHCOR″ or CH₂CONHR″; and,    -   R₆, R₇ and R₈ are independently selected from hydrogen, alkyl or        alkoxy;    -   X is selected from hydroxyl, nitro, amino, aryl, heteroaryl,        alkyl, alkoxy, CHO, COR, halogen, haloalkyl, NH₂, NHR, NRR′, SR,        CONH₂, CONHR, CONHRR′, O-aryl, O-heteroaryl, O-ester, or the        group Y—Z as defined above;    -   or a salt or derivative thereof;    -   the process comprising exposing the compound to light so that it        isomerises from the E-isomer to the Z-isomer. This process might        be carried out separately or in conjunction with the light        activated release of the compound from a prodrug as defined        above.

In a further aspect, the present invention provides a process forproducing the photoactivatable compounds defined herein, the processcomprising linking a photoactivatable group to the Y group of aprecursor compound to produce photoactivatable compounds as definedabove.

The work disclosed herein arises from the findings that the inactivetrans isomer of combretastatin A-4 E-1 can be converted to the activecis-isomer Z-1 by the action of ultraviolet light ex situ in aphotochemical reactor. The irradiation of E-1 in this manner leads to animpressive and rapid increase in activity. Further we have found thatonly after a long period of irradiation is the formation of thephenanthrene (which is only moderately active, as measured by itsability to inhibit cancer cell growth in vitro, IC₅₀ 0.7 μM) evident. Wehave prepared phenanthrene, by irradiation of E-1, in good yield when anoxidant (I₂) is present (to oxidize the first-formed cyclizationproduct). This provides an opportunity to exploit the hypoxic nature ofsolid tumours and increase the selectivity of irradiated E-1. In otherwords, healthy cells may provide an oxidative pathway for the formationof the less toxic phenanthrene and effectively decrease the lifetime ofZ-1.

The same result can be achieved in situ in the presence of culturedcancer cells (K562 human myelogenous leukaemia cell line. Theseexperiments showed that within 2 seconds of exposure to ultravioletlight the activity of the E-combretastatin A-4 (IC₅₀ originally 5 μM)increases to 2 nM, providing a rapid thousand-fold increase in activity.Moreover, the cells in the absence of the drug are not affected byexposure to the radiation and grow normally over the 5 days of theassay. To increase the water solubility of the drug we have producedprodrugs with a photo-cleavable group attached to the B-ring phenolic OHgroup. The nitro vanillin derivative was chosen since it has been usedas a photo-cleavable linker for solid phase synthesis applications andits synthesis is relatively simple. These prodrugs have beensuccessfully cleaved in both the E and Z series (E-6 and Z-6respectively) and have produced highly cytotoxic agents in vitro upon insitu exposure to ultra violet radiation. The cleavage of the watersolubilising group appears to be faster than the E→Z isomerisation, atleast under ex situ irradiation. Thus, the use of Z-6 has some merit.Indeed it forms the prototype for systems that do not rely on anyspecial photochemical features of the molecule to be delivered. Thisprovides a more general approach to the site-specific photochemicalactivation of prodrugs. Moreover, the photocleavable water solubilisinggroup can be engineered, so that cleavage occurs at longer wavelengthand rapidly.

Embodiments of the present invention will now be described by way ofexample and not limitation with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the anti-tumour activity of compound 97-64H in an in vivotumour implant experiment in mice.

FIG. 2 shows the anti-tumour activity of compound 97-96 in an in vivotumour implant experiment in mice.

DETAILED DESCRIPTION Pharmaceutical Compositions

The compounds of the invention may be derivatised in various ways. Asused herein “derivatives” of the compounds includes salts, esters suchas in vivo hydrolysable esters, free acids or bases, hydrates, prodrugsor coupling partners. In the case of compounds which are combretastatinor analogues thereof, preferably the derivatives are soluble in waterand/or saline or can be hydrolysed to provide physiologically activeagents.

Examples in the prior art of salts or prodrugs of cis-combretastatin A-4focus on forming salts or derivatives at the phenolic hydroxyl group ofcombretastatin. These include sodium phosphate salts, sodium andpotassium salts (U.S. Pat. No. 5,561,122), lithium, caesium, magnesium,calcium, manganese and zinc salts of cis-combretastatin A-4, andammonium cation salts with imidazole, morpholine, piperazine,piperidine, pyrazole, pyridine, adenosine, cinchonine, glucosamine,quinine, quinidine, tetracycline and verapamil (WO99/35150).

Salts of the compounds of the invention are preferably physiologicallywell tolerated and non toxic. Many examples of salts are known to thoseskilled in the art. Compounds having acidic groups, can form salts withalkaline or alkaline earth metals such as Na, K, Mg and Ca, and withorganic amines such as triethylamine and Tris(2-hydroxyethyl)amine.Salts can be formed between compounds with basic groups, e.g. amines,with inorganic acids such as hydrochloric acid, phosphoric acid orsulfuric acid, or organic acids such as acetic acid, citric acid,benzoic acid, fumaric acid, or tartaric acid. Compounds having bothacidic and basic groups can form internal salts.

Esters can be formed between hydroxyl or carboxylic acid groups presentin the compound and an appropriate carboxylic acid or alcohol reactionpartner, using techniques well known in the art. Examples of estersinclude those formed between the phenolic hydroxyl of the substitutedstilbenes and carboxylic acids, hemisuccinic acid esters, phosphateesters, BOC esters, sulphate esters and selenate esters.

Derivatives which as prodrugs of the compounds are convertible in vivoor in vitro into one of the parent compounds. Typically, at least one ofthe biological activities of compound will be reduced in the prodrugform of the compound, and can be activated by conversion of the prodrugto release the compound or a metabolite of it. Example of prodrugsinclude combretastatin A1 phosphate, combretastatin A4 phosphate andRH1.

Other derivatives include coupling partners of the compounds in whichthe compounds is linked to a coupling partner, e.g. by being chemicallycoupled to the compound or physically associated with it. Examples ofcoupling partners include a label or reporter molecule, a supportingsubstrate, a carrier or transport molecule, an effector, a drug, anantibody or an inhibitor. Coupling partners can be covalently linked tocompounds of the invention via an appropriate functional group on thecompound such as a hydroxyl group, a carboxyl group or an amino group.

The compounds described herein or their derivatives can be formulated inpharmaceutical compositions, and administered to patients in a varietyof forms, in particular to treat conditions which are ameliorated by theactivation of the compound.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder, cream, liquid form or encapsulated by liposomes. Atablet may include a solid carrier such as gelatin or an adjuvant or aninert diluent. Liquid pharmaceutical compositions generally include aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, or glycolssuch as ethylene glycol, propylene glycol or polyethylene glycol may beincluded. Such compositions and preparations generally contain at least0.1 wt % of the compound.

Parental administration includes administration by the following routes:intravenous, cutaneous or subcutaneous, nasal, intramuscular,intraocular, transepithelial, intraperitoneal and topical (includingdermal, ocular, rectal, nasal, inhalation and aerosol), and rectalsystemic routes. For intravenous, cutaneous or subcutaneous injection,or injection at the site of affliction, the active ingredient will be inthe form of a parenterally acceptable aqueous solution which ispyrogen-free and has suitable pH, isotonicity and stability. Those ofrelevant skill in the art are well able to prepare suitable solutionsusing, for example, solutions of the compounds or a derivative thereof,e.g. in physiological saline, a dispersion prepared with glycerol,liquid polyethylene glycol or oils.

In addition to one or more of the compounds, optionally in combinationwith other active ingredient, the compositions can comprise one or moreof a pharmaceutically acceptable excipient, carrier, buffer, stabiliser,isotonicizing agent, preservative or anti-oxidant or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material maydepend on the route of administration, e.g. orally or parentally.

Liquid pharmaceutical compositions are typically formulated to have a pHbetween about 3.0 and 9.0, more preferably between about 4.5 and 8.5 andstill more preferably between about 5.0 and 8.0. The pH of a compositioncan be maintained by the use of a buffer such as acetate, citrate,phosphate, succinate, Tris or histidine, typically employed in the rangefrom about 1 mM to 50 mM. The pH of compositions can otherwise beadjusted by using physiologically acceptable acids or bases.

Preservatives are generally included in pharmaceutical compositions toretard microbial growth, extending the shelf life of the compositionsand allowing multiple use packaging. Examples of preservatives includephenol, meta-cresol, benzyl alcohol, para-hydroxybenzoic acid and itsesters, methyl paraben, propyl paraben, benzalconium chloride andbenzethonium chloride. Preservatives are typically employed in the rangeof about 0.1 to 1.0% (w/v).

Preferably, the pharmaceutically compositions are given to an individualin a “prophylactically effective amount” or a “therapeutically effectiveamount” (as the case may be, although prophylaxis may be consideredtherapy), this being sufficient to show benefit to the individual.Typically, this will be to cause a therapeutically useful activityproviding benefit to the individual. The actual amount of the compoundsadministered, and rate and time-course of administration, will depend onthe nature and severity of the condition being treated. Prescription oftreatment, e.g. decisions on dosage etc, is within the responsibility ofgeneral practitioners and other medical doctors, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,16th edition, Osol, A. (ed), 1980. By way of example, and thecompositions are preferably administered to patients in dosages ofbetween about 0.01 and 100 mg of active compound per kg of body weight,and more preferably between about 0.5 and 10 mg/kg of body weight. Thecompounds may be used in the treatment of cancer and other conditionsinvolving abnormal proliferation of vasculature including diabeticretinopathy, psoriasis and endometriosis.

General

Proton nuclear magnetic resonance (¹H NMR) spectra were recorded on aBrüker AC 300 (300 MHz) or AC 400 (400 MHz) NMR spectrometer. Chemicalshifts, δ, for all NMR spectra are given in ppm, relative totetramethylsilane, and, unless otherwise stated, using CDCl₃ as bothsolvent and internal standard. Coupling constants (J) were measured inHz. Melting points were determined on a Gallenkamp melting pointapparatus and are uncorrected. The UV/VIS spectra were determined usinga Hewlett-Packard HP8452 diode-array spectrophotometer. Extinctioncoefficients (a) are presented as their natural logarithms.Microanalyses were carried out by the laboratories of the Departments ofChemistry of the University of Manchester and UMIST. High resolutionmass spectroscopy was determined using a Kratos Concept massspectrometer. Thin layer chromatography (tlc) was performed usingprecoated aluminium-backed silica gel plates (60 F₂₅₄) with 0.2 mmthickness (Merck), with observation under UV when necessary. Gaschromatography was carried out using an SE 54 column at 195-225 kPa at1.5 kPa/min. The oven temperature was 180-280° C. at 5° C./min.

Example 1 Synthesis of Combretastatins with Alkyl Groups on the DoubleBond Z- andE-1-(3′-t-Butyldimethylsilyloxy-4′-methoxyphenyl)-2-(3″,4″,5″-trimethoxyphenyl)propene,17a, 17b

To a slurry of 3-t-butyldimethylsilyloxy-4-methoxybenzylphosphoniumbromide, 18, (1 g, 1.69 mmol) in THF (10 ml) was added n-butyllithium(1.16 ml of 1.6 M solution, 1.86 mmol) at −15° C. The red anion wasstirred for 20 minutes and 3,4,5-trimethoxyacetophenone (355 mg, 1.69mmol) added. The resultant solution was stirred at room temperature for1 hour and water (10 ml) carefully added. The aqueous layer wasseparated and extracted with ether (3×10 ml). The combined organiclayers were washed with water (2×10 ml) and brine (10 ml), dried (MgSO₄)and concentrated in vacuo.

Following flash column chromatography (SiO₂ petrol:EtOAc 19:1) the Zstilbene, 17a, was isolated as a colourless oil (109 mg, 15%).R_(f)=0.72 (SiO₂ petrol:EtOAc 1:1); δ_(H) (300 MHz) 0.20 [6H, s,(CH₃)₂], 1.03 [9H, s, (CH₃)₃], 2.28 (3H, d, J=1.1, CH₃), 3.85 (3H, s,OCH₃), 3.89 (3H, s, OCH₃), 3.93 [6H, s, (OCH₃)₂], 6.69 (1H, q, J=1.1,olefinic H), 6.72 (2H, s, H-2′,6′) 6.87 (1H, d, J=8.3, H-5″), 6.91 (1H,d, J=2.3, H-2″), 6.95 (1H, dd, J=8.3, 2.3, H-6″); λ_(max) (MeOH)=270(s=7,607); M⁺, found 444.2329; C₂₅H₃₆O₅Si requires M⁺ 444.2332.

Further elution afforded the E stilbene, 17b, as a colourless oil (258mg, 34%). R_(f)=0.67 (SiO₂ petrol:EtOAc 1:1); δ_(H) (300 MHz) 0.01 [6H,s, (CH₃)₂], 0.92 [9H, s, (CH₃)₃], 2.17 (3H, d, J=1.5, CH₃), 3.75 [6H, s,(OCH₃)₂], 3.76 (3H, s, OCH₃), 3.87 (3H, s, OCH₃), 6.35 (1H, q, J=1.5,olefinic H), 6.42 (2H, s, H-2′,6′) 6.52 (1H, d, J=1.9, H-2″), 6.62 (1H,dd, J=8.3, 1.9, H-6″), 6.67 (1H, d, J=8.3, H-5″); λ_(max) (MeOH)=296(e=16,541). M⁺, found 444.2333; C₂₅H₃₆O₅Si requires M⁺ 444.2332.

Z-(3′-Hydroxy-4′-methoxyphenyl)-2-(3″,4″,5″-trimethoxyphenyl)propene, 19

To a stirred solution ofZ-(3′-t-butyldimethylsilyloxy-4′-methoxyphenyl)-2-(3″,4″,5″-trimethoxyphenyl)propene,17a, (111 mg, 0.250 mmol) in dry THF (5 ml) was addedtetra-n-butylammonium fluoride (700 ml of 1 M solution, 0.7 mmol). Theresulting yellow solution was stirred for 20 minutes and then treatedwith water (2 ml). The aqueous layer was separated and extracted withchloroform (3×10 ml). The combined organic layers were washed with water(2×10 ml) and brine (10 ml), dried (MgSO₄) and concentrated in vacuo.Flash column chromatography (SiO₂ petrol:EtOAc 2:1) affordedZ-(3′-hydroxy-4′-methoxyphenyl)-2-(3″,4″,5″-trimethoxyphenyl)propene,19, as a fine white powder (49 mg, 0.148 mmol, 60%). m.p. 156-8° C.;R_(f)=0.36 (SiO₂ petrol:EtOAc 2:1); δ_(H) (300 MHz) 2.18 (3H, d, J=1.5,CH₃), 3.75 [6H, s, (OCH₃)₂], 3.84 (3H, s, OCH₃), 3.88 (3H, s, OCH₃),5.42 (1H, s, OH), 6.36 (1H, q, J=1.5, olefinic H), 6.43 (2H, s, H-2′,6′)6.48 (1H, dd, J=8.3, 2.3, H-6″), 6.62 (1H, d, J=8.3, H-5″), 6.63 (1H, d,J=2.3, H-2″); λ_(max) (MeOH)=270 (e=11,524); M⁺, found 330.1469;C₁₉H₂₂O₅ requires M⁺ 330.1467.

Z-1-(3′,4′,5′-trimethoxyphenyl)-2-(3″-hydroxy-4″-methoxyphenyl)propene,20

A mixture of cis-1-(3′,4′,5′-trimethoxyphenyl)propene 21 (0.42 g, 2mmol), 5-iodo-2-methoxyphenol 22 (1 g, 4 mmol), triethylamine (0.51 g, 5mmol), palladium acetate (9 mg, 0.04 mmol) and triphenylphosphine (21mg, 0.08 mmol) were heated at 100° C. To the cooled reaction mixture wasadded aqueous hydrochloric acid (45 ml of a 2.7 M solution). Afterstirring for 10 min, the liquid was decanted off and the solid residueextracted with several portions of hot hexane. The combined hot hexanefractions were filtered. The cooled hexane solution was washed withwater (2×10 ml), brine (10 ml), dried over magnesium sulfate, filteredand the solvent evaporated. Flash column chromatography (SiO₂petrol:EtOAc 15:1) afforded the stilbene (20) as a white crystallinesolid (109 mg, 16%). m.p. 99-100° C.; R_(f)=0.34 (SiO₂ petrol:EtOAc1:1); δ_(H) (300 MHz) 2.28 (3H, d, J=1.1, CH₃), 3.89 (3H, s, OCH₃), 3.90(6H, s, 2×OCH₃), 3.94 (3H, s, OCH₃), 5.61 (1H, s, OH), 6.59 (2H, s,H-2′,6′), 6.74 (1H, q, J=1.1, olefinic H), 6.87 (1H, d, J=8.7, H-5″),7.04 (1H, dd, J=8.7, 2.3, H-6″), 7.14 (1H, d, J=2.3, H-2″). Found C,69.13; H, 6.71; C₁₉H₂₂O₅ requires C, 69.07; H, 6.71%; M⁺, found331.1541; C₁₉H₂₂O₅ (+H) requires 331.1545; λ_(max) (MeOH)=296(ε=14,126).

The ethyl derivative (45) has also been synthesised.

Z- and E-1-(4′-Methoxyphenyl)-2-(3″,4′,5″-trimethoxyphenyl)propene, 23a,23b

To a slurry of 4-methoxybenzylphosphonium chloride (598 mg, 1.43 mmol)in THF (8 ml) was added n-butyllithium (990 ml of 1.6 M solution, 1.58mmol) at −15° C. The red anion was stirred for 20 minutes and3,4,5-trimethoxyacetophenone (300 mg, 1.43 mmol) added. The resultantsolution was stirred at room temperature for 1 hour and water (10 ml)carefully added. The aqueous layer was separated and extracted withether (3×10 ml). The combined organic layers were washed with water(2×10 ml) and brine (10 ml), dried (MgSO₄) and concentrated in vacuo.

The nmr of the crude reaction product showed that the Z:E ratio was1:1.5. Following flash column chromatography (SiO₂ petrol:EtOAc 9:1) theZ stilbene, 23a, was isolated as white needles (45 mg, 0.143 mmol, 10%).m.p. 73-5° C.; R_(f)=0.46 (SiO₂ petrol:EtOAc 3:1); δ_(H) (300 MHz) 2.19(1H, d, J=1.5, CH₃), 3.74 [6H, s, (OCH₃)₂], 3.76 (3H, s, OCH₃), 3.88(3H, s, OCH₃), 6.40 (1H, q, J=1.5, olefinic H), 6.42 (2H, s, H-2′,6′),6.69 (1H, dt, J=8.7, 2.3, H-3″,5″), 6.93 (1H, dt, J=8.7, 2.3, H-2″,6″);λ_(max) (MeOH)=273 (ε=14,926).

Further elution afforded the E stilbene, 23b, as an off white solid (44mg, 0.140 mmol, 9.8%). m.p. 80-2° C.; R_(f)=0.41 (SiO₂ petrol:EtOAc3:1); δ_(H) (300 MHz) 2.28 (1H, d, J=1.5, CH₃), 3.86 (3H, s, OCH₃), 3.90(3H, s, OCH₃), 3.94 [6H, s, (OCH₃)₂], 6.74 (2H, s, H-2′,6′), 6.75 (1H,q, J=1.5, olefinic H), 6.94 (1H, dt, J=8.7, 2.3, H-3″,5″), 7.34 (1H, dt,J=8.7, 2.3, H-2″,6″); λ_(max) (MeOH)=287 (ε=21,822).

Example 2 Synthesis of Combretastatins with Alkyl Groups Replacing theMethoxy Groups on the A RingE-2-(3′,4′,5′-trimethylphenyl)-3-(3″-(2′″,3′″,5′″,6′″-tetrafluoropyridoxy)-4″-methoxyphenyl)prop-2-enoicacid, 24

A mixture of 3-(2′,3′,5′,6′-tetrafluoropyridoxy)-4-benzaldehyde 25 (2 g,6.64 mmol), 3,4,5-trimethylphenylacetic acid 26 (2.37 g, 13.3 mmol)acetic anhydride (6 ml) and triethylamine (3 ml) were heated underreflux for 3 h. After acidification with concentrated hydrochloric acid(9 ml), the solid was filtered off and recrystallised from ethanol togiveE-2-(3′,4′,5-trimethyl)-3-(3″-(2′″,3′″,5′″,6′″-tetrafluoropyridoxy)-4″-methoxyphenyl)prop-2-enoicacid 24 as a yellow crystalline solid (700 mg, 1.52 mmol, 23%). m.p.184-6° C. δ_(H) (300 MHz, DMSO) 2.11, (3H, s, CH₃), 2.14 (6H, s,(CH₃)₂), 3.83 (3H, s, OCH₃), 6.61 (1H, d, J=1.5, H-2″), 6.71 (2H, s,H-2′,6′), 7.13 (1H, d, J=8.7, H-5″), 7.19 (1H, dd, J=8.7, 1.5, H-6″),7.61, (1H, s, olefinic H), 12.52, (1H, s OH).

(Z)-1-(3′,4′,5′-trimethylphenyl)-2-(3″-(2′″,3′″,5′″,6′″-tetrafluoropyridoxy)-4″-methoxyphenyl)ethene, 27

(E)-2-(3′,4′,5′-trimethylphenyl)-3-(3″-(2′″,3′″,5 ″,6′″-tetrafluoropyridoxy)-4″-methoxyphenyl)prop-2-enoic acid 24(700 mg, 1.52 mmol) was added to powdered copper (500 mg, 7.81 mmol) inquinoline (5.5 ml, 6.02 g, 28.2 mmol) and the resulting mixture washeated at 200° C. for 2 h. Upon cooling, ether was added and the copperfiltered off through celite. The filtrate was washed with 1 Mhydrochloric acid (2×20 ml) and the aqueous layer separated andextracted with ether (3×50 ml). The combined organic layers were washedwith saturated sodium carbonate (50 ml), water (2×50 ml) and brine (50ml), dried (MgSO₄) and concentrated in vacuo. Flash columnchromatography (SiO₂ petrol:EtOAc 9:1) afforded(Z)-1-(3′,4′,5′-trimethylphenyl)-2-(3″-(2′″,3′″,5′″,6′″-tetrafluoropyridoxy)-4″-methoxyphenyl)ethene27 as a yellow oil (224 mg, 0.538 mmol, 35%). R_(f)=0.48 (SiO₂petrol:EtOAc 9:1); 86 (300 MHz) 2.15 (3H, s, CH₃), 2.19 [6H, s, (CH₃)₂],3.84 (3H, s, OCH₃), 6.41 (1H, d, J=12.1, olefinic H), 6.51 (1H, d,J=12.1, olefinic H), 6.87 (2H, s, H-2′,6′), 6.89 (1H, d, J=8.7, H-5″),6.98 (1H, d, J=2.3, H-2″), 7.11 (1H, dd, J=8.7, 2.3, H-6″).

(Z)-1-(3′,4′,5′-trimethylphenyl)-2-(3″-hydroxy-4″-methoxyphenyl)ethene,28

To a solution of the(Z)-1-(3′,4′,5′-trimethylphenyl)-2-(3″-(2′″,3′″,5′″,6′″-tetrafluoropyridoxy)-4″-methoxyphenyl)ethene(100 mg, 0.24 mmol) 27 in dry DMF (600 ml) and dichloromethane (115 ml)at 0° C. was added sodium methoxide (25 mg, 0.463 mmol). After stirringovernight, the mixture was partitioned between ether (5 ml) and 1 Msulfuric acid (5 ml). The organic phase was washed with water (5 ml),dried (MgSO₄) and concentrated in vacuo. Flash column chromatography(SiO₂ petrol:EtOAc 9:1) and recrystallisation from petrol afforded(Z)-1-(3′,4′,5′-trimethylphenyl)-2-(3″-hydroxy-4″-methoxyphenyl)etheneas a white crystalline solid (31 mg, 0.116 mmol, 48%). m.p. 60-1° C.R_(f)=0.39 (SiO₂ petrol:EtOAc 4:1); δ_(H) (300 MHz) 2.16 (3H, s, CH₃),2.21 [6H, s, (CH₃)₂], 3.90 (3H, s, OCH₃), 5.50 (1H, s, OH), 6.40 (1H, d,J=12.4, olefinic H), 6.45 (1H, d, J=12.4, olefinic H), 6.72 (1H, d,J=8.3, H-5″), 6.82 (1H, dd, J=8.3, 2.3, H-6″), 6.91 (1H, d, J=2.3,H-2″), 6.96 (2H, s, H-2′,6′).

Example 3 Synthesis of Combretastatins with a 3,4,5 Trialkoxy Group Z-andE-1-(3′,4′,5′-triethoxyphenyl)-2-(3″-t-butyldimethylsilyloxy-4″-methoxyphenyl)ethene,30a, 30b

To a slurry of 3,4,5-triethoxybenzylphosphonium bromide 29 (2 g, 3.24mmol) in THF (30 ml) was added n-butyllithium (2.5 ml of 1.6M solutionin hexanes, 4 mmol) at −15° C. under argon. The red anion was stirredfor 20 min and 3-O-t butyldimethylsilyl-4-methoxybenzaldehyde 6 (0.86 g,3.24 mmol) added. The resultant solution was stirred for 1 h at roomtemperature and water (10 ml) was carefully added. The aqueous layer wasseparated and extracted with ethyl acetate (3×100 ml). The combinedorganic layers were washed with water (2×100 ml), brine (100 ml), dried(MgSO₄) and concentrated in vacuo. Flash column chromatography affordedthe cis stilbene 30a as a colourless oil (0.23 g, 15%). R_(f)=0.65(petrol:ethyl acetate 9:1); δ_(H) (300 MHz) 0.08 (6H, s, Si(CH ₁)₂),0.95 (9H, s, 3×CH₃), 1.35 (9H, m, 3×OCH₂CH ₃), 3.79 (3H, s, OCH₃), 3.91(4H, q, J=6.8, CH₂), 4.06 (2H, q, J=7.2, CH₂), 6.40 (1H, d, J=12.1,olefinic H), 6.43 (1H, d, J=12.1, olefinic H), 6.74 (2H, s, ArH 2, 6),6.83 (1H, dd, J=8.0, 2.1, ArH para to OSi), 6.84 (1H, d, J=8.0, ArHortho to OMe), 6.88 (1H, d, J=2.1, ArH ortho to OSi).

Further elution gave the trans stilbene 30b as white crystals (0.27 g,17.6%). R_(f)=0.75; δ_(H) (300 MHz) 0.20 (6H, s, Si(CH ₃)₂), 1.03 (9H,s, 3×CH₃), 1.37 (3H, t, J=7.5, CH₂CH ₃), 1.46 (6H, t, J=7.2, 2×CH₂CH ₃),3.85 (3H, s, OCH₃), 4.12 (6H, m, 3×CH₂), 6.73 (2H, s, ArH 2, 6), 6.84(1H, dd, J=7.8, 1.98, ArH para to OSi), 6.90 (1H, d, J=15.1, olefinicH), 7.04 (1H, d, J=7.8, ArH ortho to OMe), 7.05 (1H, d, J=15.1, olefinicH), 7.66 (1H, d, J=2.0, ArH ortho to OSi).

Z- andE-1-(3′,4′,5′-triethoxyphenyl)-2-(3″-hydroxy-4″-methoxyphenyl)ethene,31a, 31b

To a stirred mixture of cis andtrans-1-(3′,4′,5′-triethoxyphenyl)-2-(3″-tert-butyldimethylsiloxy-4″-methoxy)ethene30a, 30b (0.23 g, 0.48 mmol-cis isomer; 0.27 g, 0.57 mmol-trans isomer)in dry THF (17.5 ml) was added tetra-n-butylammonium fluoride (1.46 mlof 1 M solution in THF). The resulting yellow solution was stirred fortwenty min and treated with water (50 ml). The aqueous layer wasseparated and extracted with chloroform (3×50 ml). The combined organiclayers were washed with water (2×50 ml) and brine (50 ml), dried (MgSO₄)and concentrated in vacuo.

Flash column chromatography (petrol:ethyl acetate 4:1) affordedZ-1-(3′,4′,5′-triethoxyphenyl)-2-(3″-hydroxy-4″-methoxyphenyl)ethene 31aas a colourless oil (0.08 g, 46%). R_(f)=0.24. δ_(H) (300 MHz) 1.34 (9H,m, 3×CH₂CH ₃), 3.87 (3H, s, OCH₃), 3.92 (4H, q, J=7.2, 2×CH₂), 4.06 (2H,q, J=7.2, CH₂), 6.40 (1H, d, J=12.43, olefinic H), 6.45 (1H, d, J=12.4,olefinic H), 6.50 (2H, s, ArH 2,6), 6.75 (1H, d, J=8.1, ArH ortho toOMe), 6.79 (1H, dd, J=8.7, 1.88, ArH para to OH), 6.91 (1H, d, J=1.9,ArH ortho to OH). M⁺, 358.

Further elution affordedE-1-(3′,4′,5′-triethoxyphenyl)-2-(3″-hydroxy-4″-methoxyphenyl)ethene 31b(0.09 g, 46.6%) as white crystals. mp: 91-92° C.; R_(f)=0.11. δ_(H) (300MHz) 1.37 (3H, t, J=6.0, CH₃), 1.46 (6H, t, J=6.4, 2×CH₃), 3.92 (3H, s,OCH₃), 4.12 (6H, m, 3×CH₂), 6.71 (2H, s, H 2, 6), 6.84 (1H, d, J=7.8,ArH ortho to OMe), 6.85 (1H, d, J=15.5, olefinic H), 6.91 (1H, dd,J=7.9, 2.26, ArH para to OH), 7.11 (1H, d, J=15.5, olefinic H), 7.13(1H, d, J=2.3, ArH ortho to OH). M⁺, 358.

Z- andE-1-(3′,4′,5′-triethoxyphenyl)-2-(3″-fluoro-4″-methoxyphenyl)ethene,32a, 32b

To a slurry of 3,4,5-triethoxybenzylphosphonium bromide 29 (2 g, 3.24mmol) in THF (30 ml) was added n-butyllithium (2.5 ml of 1.6 M solutionin hexanes, 4 mmol) at −15° C. under argon. The red anion was stirredfor 20 min and 3-fluoro-4-methoxybenzaldehyde (0.50 g, 3.24 mmol) wasadded. The resultant solution was stirred for 1 h and water (10 ml) wascarefully added. The aqueous layer was separated and extracted withethyl acetate (3×100 ml). The combined organic layers were washed withwater (2×100 ml) and brine (100 ml), dried (MgSO₄) and concentrated invacuo.

Flash column chromatography (SiO₂, petrol:ethyl acetate 20:1) affordedthe cis-stilbene 32a as a pale yellow oil (0.35 g, 29%), R_(f)=0.16;δ_(H) (300 MHz) 1.35 (9H, m, 3×CH₃), 3.87 (3H, s, OCH₃), 3.92 (4H, q,J=6.8, 2×CH₂), 4.07 (2H, q, J=7.2, CH₂), 6.41 (1H, d, J=12.4, olefinicH), 6.47 (1H, d, J=12.4, olefinic H), 6.47 (2H, s, ArH 2,6), 6.84 (1H,t, J=8.7, ArH ortho to OMe), 7.00 (1H, dd, J=8.7, 1.5, ArH para to F),7.05 (1H, d, J=12.1, 1.5, ArH ortho to F). M⁺, 360.

Further elution afforded the trans isomer 32b (0.27 g, 23%) as whiteneedles mp:97-99° C.; Rf=0.22; δ_(H) (300 MHz) 1.38 (6H, t, J=7.2,2×CH₃), 1.46 (3H, t, J=7.2, CH₃) 3.92 (3H, s, OCH₃), 4.12 (6H, m,3×CH₂), 6.71 (2H, s, H 2,6) 6.91 (1H, d, J=15.5, olefinic H), 6.94 (1H,t, J=8.7, ArH ortho to OMe), 6.95 (1H, d, J=15.5, olefinic H), 7.17 (1H,dd, J=8.7, 2.3 ArH para to F), 7.25 (1H, dd, J=12.0, 2.3, ArH ortho toF). M⁺, 360.

Example 4 Boc-Combretastatin Compounds

Boc-Phenylalanine Combretastatin A-4, 33

To a stirred solution of t-butoxycarbonyl-phenylalanine (168 mg, 0.634mmol), dicyclohexylcarbodiimide (157 mg, 0.76 mmol),N,N-4-dimethylaminopyridine (8 mg, 61 μmol) in dichloromethane (15 ml)under nitrogen at room temperature in the dark was added combretastatinA-4 (1) (200 mg, 0.633 mmol). After stirring for 48 h, the mixture wasfiltered, evaporated and the residue chromatographed on silica usingpetroleum (bp 40-60° C.)/ethyl acetate 4:1 to afford the title ester(33) as a clear gum (143 mg, 40%). ν_(max) 3364 (NH); 1766, 1716 (C═O).6, 7.22 (5H, m, phenylalanine ArHs); 7.11 (1H, dd, J=8.1, 2.0, H para toO ester); 6.95 (1H, d, J=2.0, H ortho to O ester); 6.83 (1H, d, J=2.0, Hmeta to O ester); 6.40, 6.50 (4H, 2 s, olefinic Hs, A-ring Hs); 5.07(1H, broad, NH); 4.80 (1H, m, H-α); 3.80, 3.70, 3.66 (12H, 3×s, 4×OMe);3.35-3.08 (2H, m, CH₂); 1.40 (9H, s, 3×CH₃). λ_(max) 241; 271; 305. m/z463 (35%, M+H—BOC); 317 (100).

The other BOC compounds 34-44 were made using the above method.

33 R = Phe 39 R = Ala 34 R = Ile 40 R = His 35 R = Gly 41 R = Pro 36 R =Trp 42 R = D-Met 37 R = Met 43 R = D-Trp 38 R = Leu 44 R = Tyr

Example 5 Benzoquinone Compounds2-Methoxy-5-[(Z)-2-(3′,4′,5′-trimethoxyphenyl)-vinyl]-[1,4]benzoquinone97-96

To a mixture of Aliquat 336 (0.181 ml, 1.25 equiv) and NaH₂PO₄.H₂O (323mg, 2.34 mmol) in water (100 ml) was added a solution of combretastatinA4 (1) (100 mg, 0.316 mmol) in dichloromethane (7 ml). Fremy's salt(potassium nitrosodisulfonate) (212 mg, 0.8 mmol) was added and themixture shaken for 30 min. (Colour changes from mauve to red). Thedichloromethane was collected and the aqueous fraction extracted withdichloromethane (3×10 ml). The combined organic phases were washed withwater (3×10 ml), brine (10 ml) and dried over magnesium sulfate.Evaporation of the solvent followed by flash chromatography of theresidue using petrol:ethyl acetate (65:35) as eluent afforded thequinone as a red crystalline solid (51 mg, 49%) mp 130-2° C., δ_(H)(acetone d6) 3.70, 3.75, 3.85 (12H, 3 s, 4×OMe); 6.08 (1H, s, quinone-Hortho to OMe); 6.43 (1H, dd, J 12.5, 0.5, olefinic-H next to quinonering); 6.67 (1H, d, J 0.5, quinone-H meta to OMe); 6.73 (2H, s, ArHs);6.96 (1H, d, J, 12.5, olefinic-H next to Ar ring). λ_(max) 296(ε12,708); 470 (2038). ν_(max) 1666, 1647 cm⁻¹. M+, 330 (40%); 315(M-Me, 60); 69 (100).

This method was also used to synthesise 98-40, 98-23, 98-33 and 98-24.

These quinones may act as prodrugs producing the active hydroquinones onreduction by enzymes.

Example 6 Biological Activity of Compounds

The following assays (1-5) were carried out as described in our paper(Woods, et al, British Journal of Cancer, 71, 705-711 (1995)). Inaddition to the cell lines described in this paper, other establishedhuman cell lines (K562, HUVEC, H460, BE, H529, and HT29) were used inthe cytotoxicity/growth inhibition assay.

(1) Inhibition of tubulin assembly. The figure quoted is theconcentration required to reduce assembly of tubulin by 50%. Tubulinassembly is monitored by light scatter/absorption at 350 nm.

(2) Competition for the colchicine binding site on tubulin. The figurequoted represents the % of ³H-colchicine bound following co-incubationof test compound and ³H-colchicine with isolated tubulin. Where esterpro-drugs were used the experiments were carried out in the presence andabsence of porcine liver esterase.

(3) Cytotoxicity/growth inhibition assay. This was carried out by theMTT assay.

(4) Permeability (shape-change) assay in endothelial cells. This wascarried out using a method based on that of Watts et al. (AnticancerRes., 17, 71-75, (1997)). This involved the diffusion of a fluorescentlylabelled dextran through a barrier of endothelial cells (HUVEC) grown toconfluence on a porous membrane. The effect of agents to alter the shapeof these cells results in an increase in the permeability of the cellsto the dye. The figure represents the increase in permeability overcontrol cultures when drug is added (30 minutes, 1 mM.).

(5) Experiments to study the anti-vascular effects of these agents werecarried out as described previously by our group. (Zhao et al, EuropeanJournal of Nuclear Medicine, 26, 231-238 (1999)). The anti-vasculareffects of the agents were monitored by either positron emissiontomography (PET) or by histological examination following treatment ofmice bearing T50:80 murine breast tumours, or H460 human lung tumours.

(6) Anti-tumour efficacy of agents was determined in xenografts of H460human lung cancer. Animals (n=5) were treated either with 5 dailyinjections and tumour size measured with time following treatment.

(7) The pharmacokinetic profile of agents was determined in micefollowing injection with drug. Blood was taken at various timesfollowing treatment and analysed by HPLC with UV detection. The HPLCconditions were isocratic using a 5 micron C18 BDS column (150×4.6) andeluting with 75% MeOH in water with a detection wavelength of 290 nm.The retention time of 6.6 mins for 97-64H.

(8) Activation of quinone prodrugs by DT-diaphorase. (NAD(P)H:quinoneoxidoreductase, EC 1.6.99.2). DT-diaphorase is an enzyme over-expressedin a number of human tumours. We have recently shown this enzyme to behighly expressed in the endothelial cells of blood vessels. Thistwo-electron reducing enzyme can be used to selectively activateprodrugs within cancers which over-express the enzyme. We have thereforesynthesised quinone pro-drugs (97-96, 98-40, 98-23, 98-33, 98-24) which,upon reduction by the enzyme, produces an agent (a hydroquinonestilbene) which is cytotoxic.

Results

The in vitro studies reported in Table 11 illustrate the structuralrequirements necessary for biological activity. Modifications of the Aring have shown that the methoxy groups can be replaced by alkoxy oralkyl (28,31a,32a) whilst retaining their ability to inhibit assembly ofisolated tubulin. Similarly, molecules with alkyl substituents on theethene bridge (19,20,46) retain activity both as inhibitors of tubulinassembly and are growth inhibitory in vitro. The pharmacophore consistsof a stilbene in a cis configuration with a small alkyl or O-alkylsubstituent at the 4-position of the B ring. Substitution at the3-position of the B-ring with F results in highly active compounds thatare potent inhibitors of tubulin assembly, and are potently growthinhibitory (97-64H, and 98-35). These agents show good activity in theshape-change (permeability) assay. This test is used as an in vitroassay of vascular damage. 97-64H also shows anti-tumour activity in vivoin H460 human lung cancer xenografts. 97-64H was given at either ⅔ ofthe maximum tolerated dose (MTD=200 mg/kg) or at % of the MTD to micebearing liver metastatic T50:80 tumours. Tumours were removed andexamined for evidence of vascular damage at 2 hr and 4 hr following drugadministration. Similarly a dose equivalent to of the MTD wasadministered and the tumours examined 24 hr and 48 hr followingadministration of drug. Examination of these tumours showed tumournecrosis, blocked vessels, infiltration with red blood cells, consistentwith damage to the vasculature. These effects are seen within 2 hours.97-64H is orally bio-available when administered at 200 mg/kg in 5%dimethylacetamide in arachis oil, 97-64H showed a Cmax of 1.49 ug/ml, anabsorption half life 15 mins, and an elimination half life 32 mins. Theconcentration achieved in vivo (1.49 mg/ml=4.6 mM) is far in excess ofthe concentration necessary to inhibit the growth of HUVEC cells invitro (0.001 mM) indicating that this agent is bio-available whenadministered orally.

The 3-position of the B-ring can also be substituted with larger groupssuch as boc-amino acid esters, pyridyl esters, and ethers. The aminoacid esters (33-44) are prodrugs which, upon the action of esterase,release the active agent. The activity of these agents is related to therate of hydrolysis of these agents by esterase. The most activecompounds being those which are most readily hydrolysed. The activity ofthese compounds indicates that ester linked polymers and peptides wouldbe good prodrugs for agents of this type (see Table 1).

The pyridyl esters (96-167 and 97-07) are potent inhibitors of tubulinassembly and can displace ³H-colchicine from tubulin without the actionof esterase, showing that the 3-position of the B-ring can besubstituted with bulky side groups. Potent growth inhibitory activityand good activity in the shape-change assay can also be seen for thetetrafluoropyridyl ether (97-13) and other ethers (98-29) which are nota substrate for esterase.

A series of prodrugs capable of being activated by DT-diaphorase, anenzyme over-expressed in a wide range of human cancers and inendothelial cells of the vasculature were synthesised. The rationale forthis is that these agents will be activated in situ by the tumoursover-expressing the enzyme, thus giving rise to a high localconcentration of active drug. This confers selectivity to this agent.These quinone prodrugs (97-96 and 98-23) were tested for their abilityto act as substrates for DT-diaphorase. It can be seen that thesecompounds are good substrates for the enzyme, and are comparable to RH1(2,5,-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone) a novelalkylating agent which is activated by DT-diaphorase and is currentlyabout to enter clinical trial. An analysis of the growth inhibitoryproperties of these agents shows that 97-96 is 16-fold more active inthe H460 human lung cell line which expresses active DT-diaphorase thanin the diaphorase null H596 cell line 97-96 is active in an H460 humanlung tumour xenograft, a tumour which expresses active DT-diaphorase.This agent may therefore have a role in the treatment of tumours thatover-express DT-diaphorase. Similarly, these agents maybe selectivelyactivated in the endothelial cells of the vasculature, therebyconferring a selective anti-vascular effect.

TABLE 1 Colchicine Inhibition Colchicine displacement IC₅₀ ofdisplacement protein:drug IC₅₀ (nM) tubulin protein:drug 1:10 (nM)A2780/ assembly 1:10 (with (without Compound A2780 ADR (IC₅₀ μM)esterase) esterase) 1 0.72 0.84 2.4 NA 12 33 2.8 4.0 3.6* 2 9234 >60 >60 6.2* 2 65 35 4.3 5.0 7.5* 4 65 36 1.7 2.0 4.0* ND ND 37 6.79.1 6.0* 2 88 38 14.0 27.0 7.8* ND 72 39 2.7 4.6 8.0 2 77 40 2.2 4.37.0* 4  2 41 29.0 39.0 9.0* 2 78 42 2.9 2.9 3.5* 2 ND 43 3.3 3.8 3.0* 2ND 44 4.5 6.4 ND ND ND

TABLE 2 Compound P388 A2780 H460 H596 H596/H460 A-4 2.6 0.72 1.51 97-96570 190 38 620 16 98-40 >5000 4630 >5000 2190 <0.44 98-23 2180 2150 43702280 0.52 98-33 <780 950 4110 1490 0.36 98-24 17050 1630 3080 1950 0.63

IC₅₀ are in nM.

97-96 R═OMe (cis) 98-24

98-40 R═OMe (trans)

98-23 R=Me (cis)

98-33 R=Me (trans)

Example 7 Synthesis of Combrestatatin A-4 Bearing a Photocleavable GroupMethyl 4-(4′-formyl-2′-methoxyphenoxy)butanoate 4

(see D. L. McMinn and M. M. Greenberg, Tetrahedron, 1996, 52, 3827)

To a stirred solution of vanillin (3)(2 g, 13.16 mmol) and methyl4-bromobutyrate (2.38 g, 13.16 mmol) in DMF (20 cm³) was added freshlyground anhydrous potassium carbonate (2.03 g, 14.47 mmol). The resultingpale pink suspension was heated at 100° C. for 90 mins, after which timethe suspension has become milky pink in appearance. The mixture was thenpoured into water (50 cm³) and extracted with Et₂O (3×30 cm³). Thecombined organic extracts were washed with water (2×30 cm³), then 1M HCl(2×30 cm³) before being dried (MgSO₄) and concentrated in vacuo. Thepale pink solid 4 was used without further purification (2.7 g, 82%).m.p. 69° C. [lit. (D. L. McMinn and M. M. Greenberg, Tetrahedron, 1996,52, 3827) m.p. 68-69° C.]. Found C, 62.0; H, 6.3; C₁₃H₁₆O₅ requires C,62.0; H, 6.4%; R_(f) 0.44 (SiO₂, hexane:EtOAc, 2:1 v/v); ν_(max) (KBrdisc)/cm⁻¹ 3100-2700 (m), 1740 (s), 1680 (s); δ_(H) (300 MHz, CDCl₃)2.19 (2H, q, J=6.6 Hz, CH₂), 2.56 (2H, t, J=6.6 Hz, CH₂), 3.68 (3H, s,OCH₃), 3.91 (3H, s, OCH₃), 4.15 (2H, t, J=6.6 Hz, CH₂), 6.97 (1H, d,J=8.0 Hz, H-6′), 7.40 (1H, dd, J=8.0 Hz, J=1.8 Hz, H-5′), 7.44 (1H, d,J=1.8 Hz, H-3′), 9.84 (1H, s, CHO) ppm; δ_(C) (100 MHz, CDCl₃) 24.1(CH), 30.2 (CH₂), 51.5 (OCH₃), 55.8 (OCH₃), 67.3 (CH), 109.2 (CH), 111.5(CH), 126.6 (CH), 130.0, 149.7, 153.7, 173.3 (COOCH₃), 190.8 (CHO) ppm;m/z (FAB) 253 [(MH⁺), 50%].

Methyl 4-(4′-formyl-2′-methoxy-5′-nitrophenoxy)butanoate 5

(see D. L. McMinn and M. M. Greenberg, Tetrahedron, 1996, 52, 3827.)

To a pale pink solution of methyl4-(4′-formyl-2′-methoxyphenoxy)butanoate (4) (3 g, 12.3 mmol) in DCM (20cm³) was added dropwise, at 0° C. fuming nitric acid (1.5 cm³, 37.0mmol). The resulting green solution was stirred at 00° C. for a further30 mins before being allowed to spontaneously warm to r.t., where itremained for 3 hours. The subsequent bright yellow suspension was pouredonto iced water (50 cm³) and extracted with DCM (3×30 cm³). The combinedorganic phase was washed with sat. NaHCO₃ solution (2×50 cm³), followedby water (2×50 cm³) before being dried and concentrated in vacuoproviding a bright yellow solid. The desired compound 5 was used withoutfurther purification (2.48 g, 68%). A small amount of material (250 mg,0.84 mmol) was purified via column chromatography (SiO₂, CH₂Cl₂) so thatcomplete characterisation data could be obtained, m.p. 75° C. [lit. (D.L. McMinn and M. M. Greenberg, Tetrahedron, 1996, 52, 3827.) m.p. 76-78°C.]. Found C, 52.7; H, 5.2; N, 4.8; C₁₃H₁₅NO₇ requires C, 52.5; H, 5.1;N, 4.7%; R_(f) 0.44 (SiO₂, CH₂Cl₂); ν_(max) (KBr disc)/cm⁻¹ 3100-2700(m), 1735 (s), 1690 (s), 1290, 1220; 6, (200 MHz, CDCl₃) 2.23 (2H, q,J=7.1 Hz, CH₂), 2.57 (2H, t, J=7.1 Hz, CH₂), 3.71 (3H, s, OCH₃), 3.99(3H, s, OCH₃), 4.21 (2H, t, J=7.1 Hz, CH₂), 7.40 (1H, s, H-3′), 7.61(1H, s, H-6′), 10.44 (1H, s, CHO) ppm; δ_(C) (100 MHz, CDCl₃) 24.1(CH₂), 30.2 (CH₂), 51.8 (OCH₃), 56.6 (OCH₃), 59.6 (CH₂), 108.1 (CH),109.9 (CH), 125.5, 143.8, 151.7, 153.4, 173.2 (COOCH₃), 187.8 (CHO) ppm;m/z (FAB) 298 [(MH⁺), 40%].

Methyl 4-[4′-(hydroxymethyl)-2′-methoxy-5′-nitrophenoxy]butanoate 6

(see D. L. McMinn and M. M. Greenberg, Tetrahedron, 1996, 52, 3827.)

To a clear yellow solution of methyl4-(4′-formyl-2′-methoxy-5′-nitrophenoxy) butanoate (5) (1 g, 3.36 mmol)in THF (10 cm³) was added, portion wise, sodium borohydride (128 mg,3.36 mmol). The solution quickly changed appearance, becoming deeporange after about 5 mins. The mixture was stirred for a further 55 minsbefore water was added (25 cm³), the subsequent yellow mixture wasextracted with ether (3×30 cm³) and the combined organic extracts weredried (MgSO₄) and concentrated in vacuo providing a pale yellow solid.The solid, 6 was used without further purification (875 mg, 87.5%). Asmall amount of material (200 mg, 0.67 mmol) was purified via columnchromatography (SiO₂, hexane:EtOAc 2:1 v/v) so that completecharacterisation data could be obtained. m.p. 100° C. [lit. (D. L.McMinn and M. M. Greenberg, Tetrahedron, 1996, 52, 3827.) m.p. 98-100°C.]. Found C, 52.2; H, 5.7; N, 4.7; C₁₃H₁₇NO₇ requires C, 52.2; H, 5.7;N, 4.7%; R_(f) 0.24 (SiO₂, hexane:EtOAc 2:1 v/v); ν_(max) (KBrdisc)/cm⁻¹ 3400-3100 (br), 3000-2800 (m), 1730 (s), 1280, 1220; δ_(H)(200 MHz, CDCl₃) 2.20 (2H, q, J=7.3 Hz, CH₂), 2.56 (2H, t, J 7.3 Hz,CH₂), 2.61 (1H, br, OH), 3.70 (3H, s, OCH₃), 3.98 (3H, s, OCH₃), 4.13(2H, t, J=7.3 Hz, CH₂), 4.95 (2H, s, CH₂), 7.15 (1H, s, H-3′), 7.71 (1H,s, H-6′) ppm; δ_(C) (100 MHz, CDCl₃) 24.3 (CH₂), 30.4 (CH₂), 51.7(OCH₃), 56.4 (OCH₃), 62.8 (CH₂), 68.3 (CH₂), 109.5 (CH), 111.2 (CH),132.4, 139.6, 147.2, 154.3, 173.4 (COOCH₃) ppm; m/z (FAB) 299 [(M⁺),15%], 282 [(M-OH), 40%].

Methyl 4-[4′-(bromomethyl)-2′-methoxy-5′-nitrophenoxy]butanoate 7

To a solution of methyl4-[4′-(hydroxymethyl)-2′-methoxy-5′-nitrophenoxy]butanoate (6) (750 mg,2.52 mmol) in anhydrous THF (10 cm³) was added PBr₃ (0.24 cm³, 2.52mmol). The resulting deep yellow solution was refluxed for 3 hours,after which time no visible changes had occurred. The reaction mixturewas cooled then poured onto ice. The resulting aqueous solution wasextracted with ether (3×30 cm³). The combined organic extracts werewashed with 5% NaHCO₃ solution (2×30 cm³) and water (2×30 cm³) beforebeing dried (MgSO₄) and concentrated in vacuo. The desired compound 7was furnished as a yellow oil. The oil was then purified via columnchromatography (SiO₂, hexane:EtOAc, 2:1 v/v) thus providing 7 as ayellow powder (580 mg, 64%). m.p. 68-70° C. Found C, 43.2; H, 4.5; N,4.0; Br, 21.9; C₁₃H₁₆NO₆Br requires C, 43.1; H, 4.4; N, 3.9; Br, 22.1%;R_(f) 0.63 (SiO₂, hexane:EtOAc 2:1 v/v); ν_(max) (KBr disc)/cm⁻¹3000-2800 (m), 1720 (s), 1610, 1580, 1530, 1280, 1220; δ_(H) (200 MHz,CDCl₃) 2.20 (2H, q, J=6.7 Hz, CH₂), 2.56 (2H, t, J=6.7 Hz, CH₂), 3.70(3H, s, OCH₃), 3.97 (3H, s, OCH₃), 4.13 (2H, t, J=6.7 Hz, CH₂), 4.86(2H, s, CH₂), 6.92 (1H, s, H-3′), 7.67 (1H, s, H-6′) ppm; δ_(C) (100MHz, CDCl₃) 24.1 (CH₂), 30.1 (CH₂), 51.6 (OCH₃), 56.4 (OCH₃), 68.0(CH₂), 68.3 (CH₂), 109.7 (CH), 113.8 (CH), 127.3, 140.0, 148.1, 153.5,173.2 (COOCH₃) ppm; m/z (FAB) 362 [(MH⁺), 100%].

Methyl 4-[2′-methoxy-5′-nitro-4′-(phenoxymethyl)phenoxy]butanoate 9

To a suspension of phenol (130 mg, 138 mmol) and methyl4-[4′-(bromomethyl)-2′-methoxy-5′-nitrophenoxy]butanoate (7) (500 mg,138 mmol) in anhydrous methanol (5 cm³) was added K^(t)OBu (186 mg, 166mmol). The resulting green mixture was stirred at r.t. for 30 mins afterwhich time a white precipitate had formed. The precipitate was filteredand recrystallised from methanol affording the desired compound 9 as apure white solid (319 mg, 61%). m.p. 96-98° C. Found C, 61.0; H, 5.8; N,4.0; C₁₉H₂₁NO₇ requires C, 60.8; H, 5.6; N, 3.7%; R_(f) 0.64 (SiO₂,hexane:EtOAc 2:1 v/v); ν_(max) (KBr disc)/cm⁻¹ 3000-2800 (m), 1720 (s),1610, 1580, 1520, 1280, 1220; δ_(H) (300 MHz, CDCl₃) 2.21 (2H, q, J=6.7Hz, CH₂), 2.57 (2H, t, J=6.7 Hz, CH₂), 3.71 (3H, s, OCH₃), 3.91 (3H, s,OCH₃), 4.14 (2H, t, J=6.7 Hz, CH₂), 5.50 (2H, s, CH₂), 6.98-7.02 (3H, m,ar), 7.29-7.34 (3H, m, ar), 7.77 (1H, s, ar) ppm; δ_(C) (100 MHz, CDCl₃)24.7 (CH₂), 30.4 (CH₂), 51.7 (OCH₃), 56.3 (OCH₃), 67.0 (CH₂), 68.2(CH₂), 109.4 (CH), 109.6 (CH), 115.0 (CH), 121.5 (CH), 129.6 (CH), 129.7(CH), 138.9, 147.0, 154.3, 158.1, 173.2 (COOCH₃) ppm; m/z (FAB) 376[(MH), 20%], 282 [(M-C₆H₅O), 80%].

Methyl4-[2′-methoxy-4′-({2″-methoxy-5″-[(Z)-2′″-3″,4′″,5′″-trimethoxyphenyl)ethenyl]phenoxy}-methyl)-5′-nitrophenoxy]butanoateZ-8

To a suspension of methyl4-[4′-(bromomethyl)-2′-methoxy-5′-nitrophenoxy]butanoate (7) (1.5 g,4.14 mmol) and cis-CA-4 (1.6 g, 5.0 mmol) in anhydrous methanol (20 cm³)was added K^(t)OBu (0.58 g, 5.2 mmol). The resulting yellow mixture wasstirred at r.t. for 30 mins after which time a yellow precipitate hadformed. The precipitate was filtered and recrystallised from methanolfurnishing the desired compound Z-8 as a pale yellow solid (1.62 g,69%). m.p. 132-134° C. Found C, 65.2; H, 6.5; N, 2.4; C₃₁H₃₅NO₁₁requires C, 65.6; H, 6.2; N, 2.5%; Accurate mass; found M⁺597.2201;C₃₁H₃₅NO₁₁ requires M⁺597.2210; R_(f) 0.60 (SiO₂, hexane:EtOAc 2:1 v/v);ν_(max) (KBr disc)/cm⁻¹ 3000-2800 (m), 1730 (s), 1610, 1580, 1520, 1320,1280, 1220, 1130, 880; λ_(max) (MeCN)/nm 222 (ε35050), 242 (ε29263) and293 (ε14071); δ_(H) (300 MHz, CDCl₃) 2.21 (2H, q, J=6.7 Hz, CH), 2.58(2H, t, J=6.7 Hz, CH₂), 3.69 (6H, s, 2×OCH₃), 3.71 (3H, s, OCH₃), 3.83(3H, s, OCH₃), 3.89 (3H, s, OCH₃), 3.95 (3H, s, OCH₃), 4.51 (2H, t,J=6.7 Hz, CH₂), 5.41 (2H, s, CH₂), 6.45 (1H, d, J=12.4 Hz, olefinic H),6.49 (1H, d, J=12.4 Hz, olefinic H), 6.50 (2H, s, 2′″-H and 6′″-H), 6.82(1H, d, J=8.3 Hz, H-3″), 6.93-6.97 (2H, m, H-4″ and H-6″), 7.46 (1H, s,H-3′), 7.75 (1H, s, H-6′) ppm; δ_(C) (100 MHz, CDCl₃) 24.3 (CH₂), 30.4(CH₂), 51.7 (OCH₃), 55.9 (OCH₃), 56.0 (OCH₃), 56.2 (OCH₃), 60.9 (OCH₃),68.2 (CH₂), 68.4 (CH₂), 105.9 (CH), 109.3 (CH), 109.7 (CH), 111.6 (CH),115.6 (CH), 123.0 (CH), 129.2 (CH), 129.4 (CH), 129.6, 130.3, 132.6,137.2, 138.8, 147.0, 147.3, 149.0, 152.9, 154.3, 173.4 (COOCH₃) ppm; m/z(FAB) 376 [(M⁺), 40%].

4-[2′-methoxy-4′-({2″-methoxy-5″-[(Z)-2′″-3′″,4′″,5′″-trimethoxyphenyl)ethenyl]phenoxy}methyl)-5′-nitrophenoxy]butanoicacid Z-11

In a foil wrapped flask a suspension of the methyl ester Z-8 (250 mg,0.42 mmol) in 1M aqueous NaOH (0.84 ml, 0.84 mmol) was prepared. Theyellow suspension was then heated at reflux for 40 mins, after whichtime water (5 cm³) was added. The resulting orange solution was thenacidified to pH 1 using cone. HCl and the subsequent yellow precipitatewas filtered. The precipitate was purified by recrystallisation fromethanol, which provided the desired compound as a pale yellow solid (214mg, 81%). m.p. 138° C. Found C, 61.6; H, 5.4; N, 2.3; C₃₀H₃₃NO₁₁requires C, 61.7; H, 5.7; N, 2.4%; R_(f) 0.10 (SiO₂, hexane:EtOAc 1:1v/v); ν_(max) (KBr disc)/cm⁻¹ 3500-3100 (br), 3000-2800 (m), 1740 (s),1610, 1580, 1520, 1330, 1280, 1220, 1130, 880; δ_(H) (300 MHz, CDCl₃)2.21 (2H, q, J=6.5 Hz, CH₂), 2.63 (2H, t, J=6.5 Hz, CH₂), 3.67 [6H, s,(OCH₃)₂), 3.81 (3H, s, OCH₃), 3.88 (3H, s, OCH₃), 3.93 (3H, s, OCH₃),4.16 (2H, t, J=6.5 Hz, CH₂), 5.40 (2H, s, CH₂), 6.43 (1H, d, J=12.9 Hz,olefinic CH), 6.47 (2H, s, H-2′″ and H-6′″), 6.48 (1H, d, J=12.9 Hz,olefinic H), 6.81 (1H, d, J=7.9 Hz, H-3″), 6.91-6.95 (2H, m, H-4″ andH-6″), 7.45 (1H, s, H-3′), 7.74 (1H, s, H-6′) ppm; δ_(C) (100 MHz,CDCl₃) 24.0 (CH₂), 30.1 (CH), 55.9 (OCH₃), 56.0 (OCH₃), 56.2 (OCH₃),60.9 (OCH₃), 68.0 (CH₂), 68.4 (CH₂), 105.9 (CH), 109.4 (CH), 109.7 (CH),111.6 (CH), 115.6 (CH), 123.1 (CH), 129.2 (CH), 129.3 (CH), 129.7,130.3, 132.7, 137.2, 138.8, 146.9, 147.3, 149.0, 152.9, 154.3, 173.5(COOCH₃) ppm; m/z (FAB) 583 [(M⁺), 100%].

Methyl4-[2′-methoxy-4′-({2″-methoxy-5″-[(E)-2′″-3′″,4′″,5′″-trimethoxyphenyl)ethenyl]phenoxy}-methyl)-5′-nitrophenoxy]butanoateE-8

To a suspension ofmethyl-4-[4′-(bromomethyl)-2′-methoxy-5′-nitrophenoxy]butanoate 7 (400mg, 1.1 mmol) and trans-CA-4 (280 mg, 0.89 mmol) in anhydrous methanol(10 cm³) was added K^(t)OBu (148 mg, 1.32 mmol). The resulting yellowmixture was stirred at r.t. for 30 mins after which time a yellowprecipitate had formed. The precipitate was filtered and recrystallisedfrom methanol furnishing the desired compound E-8 as a pale yellow solid(367 mg, 73%). m.p. 142° C. Found C, 65.5; H, 6.4; N, 2.4; C₃₁H₃₅NO₁₁requires C, 65.6; H, 6.2; N, 2.5%; R_(f) 0.44 (SiO₂, hexane:EtOAc 1:1v/v); ν_(max) (KBr disc)/cm⁻¹ 3000-2800 (m), 1730 (s), 1610, 1580, 1520,1320, 1280, 1220, 1130, 880; λ_(max) (MeCN)/nm 219 (ε36612), 243(ε33655) and 329 (ε41112); δ_(H) (300 MHz, CDCl₃) 2.20 (2H, p, J=6.8 Hz,CH₂), 2.57 (2H, t, J=6.8 Hz, CH₂), 3.70 (3H, s, OCH₃), 3.86 (3H, s,OCH₃), 3.92 [6H, s, (OCH₃)₂], 3.93 (3H, s, OCH₃), 3.97 (3H, s, OCH₃),4.41 (2H, t, J=6.8 Hz, CH₂), 5.61 (2H, s, CH₂), 6.73 (2H, s, H-2′″ andH-6′″), 6.87 (1H, d, J=16.2 Hz, olefinic H), 6.94 (1H, s, H-3″), 6.95(1H, d, J_(H) 16.2 Hz, olefinic CH), 7.11-7.14 (2H, m, H-4″ and H-6″),7.55 (1H, s, H-3′), 7.77 (1H, s, H-6′) ppm; δ_(C) (100 MHz, CDCl₃) 24.4(CH₂), 30.4 (CH₂), 51.7 (OCH₃), 56.1 [(OCH₃)₂], 56.2 (OCH₃), 56.3(OCH₃), 61.0 (OCH₃), 68.4 (2×CH₂), 103.3 (CH), 109.3 (CH), 109.7 (CH),112.0 (2×CH), 120.7 (CH), 127.2 (CH), 127.5 (CH), 129.9, 130.7, 133.1,137.8, 138.8, 147.0, 147.8, 149.5, 153.4, 154.5, 173.3 (COOCH₃) ppm; m/z(FAB) 597 [(M⁺), 25%].

4-[2′-methoxy-4′-({2″-methoxy-5″-[(E)-2′″-3′″,4′″,5′″-trimethoxyphenyl)ethenyl]phenoxy}methyl)-5′-nitrophenoxy]butanoicacid E-11

In a foil wrapped flask a suspension of the methyl ester E-8 (250 mg,0.42 mmol) in 1M aqueous NaOH (0.84 ml, 0.84 mmol) was prepared. Theyellow suspension was then heated at reflux for 40 mins, after whichtime water (5 cm³) was added. The resulting orange solution was thenacidified to pH 1 using conc. HCl and the subsequent yellow precipitatewas filtered. The precipitate was purified by recrystallisation fromethanol, which provided the desired compound as a pale yellow solid (234mg, 97%). m.p. 177° C. Found C, 62.0; H, 5.8; N, 2.5; C₃₀H₃₃NO₁₁requires C, 61.7; H, 5.7; N, 2.4%; R_(f) 0.15 (SiO₂, hexane:EtOAc 1:1v/v); ν_(max) (KBr disc)/cm⁻¹ 3500-3100 (br), 3000-2800 (m), 1710 (m),1610, 1580, 1520, 1330, 1280, 1220, 1130; δ_(H) (300 MHz, CDCl₃) 2.25(2H, q, J=6.7 Hz, CH₂), 2.66 (2H, t, J=6.7 Hz, CH₂), 3.90 (3H, s, OCH₃),3.95 (3H, s, 2×OCH₃), 3.96 (3H, s, OCH₃), 4.00 (3H, s, OCH₃), 4.19 (2H,t, J=6.7 Hz, CH₂), 5.64 (2H, s, CH), 6.76 (2H, s, H-2′″ and H-6′″),6.93-6.98 (3H, m), 7.14-7.16, (2H, m, H-4″ and H-6″), 7.59 (1H, s,H-3′), 7.82 (1H, s, H-6′) ppm; δ_(C) (100 MHz, CDCl₃) 24.0 (CH₂), 30.0(CH₂), 56.1 (2×OCH₃), 56.2 (OCH₃), 56.3 (OCH₃), 61.0 (OCH), 68.1 (CH₂),68.2 (CH₂), 103.3 (2×CH), 109.3 (CH), 109.8 (CH), 112.0 (CH), 120.7(CH), 127.2 (CH), 127.5 (CH), 130.1, 130.7, 133.1, 137.9 139.2, 146.8,147.8, 149.5, 153.4, 154.8, 177.4 (COOH) ppm; m/z (FAB) 583 [(M⁺), 70%].

4-[2′-methoxy-4′-({2″-methoxy-5″-[(E)-2′″-3′″,4′″,5′″-trimethoxyphenyl)ethenyl]phenoxy}methyl)-5′-nitrophenoxy]butanoicacid (potassium salt) E-12

To a suspension of the butanoic acid E-11 (250 mg, 0.43 mmol) inmethanol (3 cm³) was added K^(t)OBu [0.43 cm³, 0.43 mmol, (1 Mmethanolic solution)]. The resulting clear yellow solution was stirredat r.t. for 5 minutes before being concentrated in vacuo to provide apale brown solid (0.26 mg, 97%).

Example 8 Photochemical Cleavage and Isomerisation Methyl4-[2′-methoxy-4′-({2″-methoxy-5″-[(E)-2′-3′″,4′″,5′″-trimethoxyphenyl)ethenyl]phenoxy}methyl)-5′-nitrophenoxy]butanoate,E-8

In a 400 cm³ photochemical reaction vessel, N₂ was bubbled throughdistilled benzene (300 cm³) for 30 mins. Following the degassingprocedure, compound E-12 (300 mg, 0.5 mmol) was added to the benzene andallowed to dissolve. The colourless solution was then irradiated using a400 W medium pressure Hg lamp for a total of 20 mins. Aliquots wereremoved from the reaction vessel at specific time points throughout themins (0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5,20.0 mins). Two aliquots were removed from the reaction vessel at eachtime point, one sample (0.5 cm³) was used for HPLC analysis (cyanopropylcolumn; mobile phase 95:5 hexane:IPA; flow rate 0.5 ml min⁻¹; lamp 245nm), whilst the other sample (10 cm³) was concentrated in vacuo and usedto determine the IC₅₀ at that time point. After irradiation for 20 minsthe reaction mixture was yellow/orange in colour.

The following compound retention times were observed:

Trans-CA-4 23.35 mins Cis-CA-4 11.07 mins Coupled trans-CA-4 E-8 43.89mins Coupled cis-CA-4 E-8 26.42 mins

Example 9 Photochemical Isomerization of Combrestatatin A-4 andDerivatives

We detail the study of the trans to cis isomerisation of CA-4 herein. Asolution of trans-CA-4 (200 mg, 0.63 mmol) in freshly distilled anddegassed benzene (400 cm³), prepared by bubbling through Ar for 2 hoursprior to use, was irradiated using a 400 W medium pressure Hg lamp for atotal of 20 mins. Aliquots were removed at regular intervals for GCanalysis (180-280° C. at 5° C. min⁻¹; pressure program 195-225 kPa at1.5 kPa min⁻¹; on a SE54 column).

The results of GC analysis of the mixture are summarized in Table 3.Table 4 shows the retention times, by GC for the individual componentsof the reaction mixture.

TABLE 3 Time (mins) % trans-stilbene % cis-stilbene % phenanthrene 0 1000 0 0.5 100 0 0 1.5 100 0 0 3.0 87 13 0 5.0 69 31 0 6.5 55 45 0 8.0 5347 0 10.0 44 53 0 12.5 32 68 0 15.0 29 71 0 17.5 22 74 4 20.0 23 73 4

TABLE 4 Component Retention time (mins)* trans-CA-4 20.32 cis-CA-4 15.42CA-4 phenanthrene derivative 1 19.00 *G.C. conditions; 180-280° C. @ 5°C. min⁻¹, 195-225 kPa @ 1.5 kPa min⁻¹, SE 54 column.

Table 5 shows the outcome of photoisomerisation of trans-CA-4 (99.7% E)and the corresponding IC₅₀ values. The cytotoxicity (IC₅₀ value) of thereaction mixture at each time point is shown in Table 3, showing animpressive increase in the cytotoxicity of the mixture over time.

The most exciting observation is that the IC₅₀ at time zero is 4000 nMand after just half a minute it has decreased by more than 10 fold. Thisillustrates that the isomerization is rapid and also highlights just howpotent cis-CA-4 is compared with trans-CA-4 since just 2.0% of cis-CA-4present results in such a dramatic increase in cytotoxicity. Thethousand-fold increase in activity obtained after six minutes, clearlyshows that the process displays great potential.

TABLE 5 Time (mins) % trans % cis % phenanthrene IC₅₀ (K562) (nM) 0 99.70.3 0 4000 0.5 98.0 2.0 0 310 1.0 96.1 3.9 0 530 1.5 93.7 6.3 0 50 2.092.7 7.3 0 20 3.0 74.3 25.7 0 12 4.0 62.3 37.3 0 9 5.0 52.1 47.9 0 107.5 40.2 56.6 1.4 3 10.0 34.0 63.1 2.9 6 12.5 32.2 64.4 3.4 4 15.0 31.764.6 3.7 1 17.5 30.8 65.1 4.1 3 20.0 30.6 65.7 3.7 2

Previously, the isomerisation study had been performed ex situ, i.e. ina photochemical reactor. However, it was necessary to illustrate thepotential of the process as a real therapy by repeating the isomerizatonin the presence of the cancer cells i.e. in situ. Therefore, K562 cellswere dosed with a known concentration of trans-CA-4, the resultingsolutions were then irradiated using an ultra violet lamp consisting of2×7 W ultra violet tubes for given lengths of time. Followingirradiation, the cells were incubated in the normal way and the IC₅₀values were determined. A series of control experiments were performedsimultaneously to verify that the results obtained (illustrated in Table6) were in fact due to the isomerisation process occurring and not dueto the effect of the ultraviolet radiation. Table 6 shows that the samepattern of results is obtained. However, the decrease in the IC₅₀ valueis now more rapid.

The experiment convincingly supports the results derived from the exsitu study and we are able to show that the trans to cis isomerisationis occurring in both circumstances, and more importantly is causing arapid and significant increase in the cytotoxicity of the system. Due tothe speed with which the process was occurring the experiment wasrepeated and monitored every 5 seconds in the first minute ofirradiation. The results are shown in Table 7. It can be seen that afterjust 5 seconds there is a greater than 15 fold reduction in the IC₅₀ andafter a further 40 seconds a single nanomolar figure IC₅₀ value isreached.

TABLE 6 Time/mins IC₅₀ K562 nM 0 1200 1 3.6 2 2.9 3 1.7 4 4.4 5 2.7 62.9 7 1.9 8 3.0 9 2.5 10 2.6 12.5 2.4 15.0 2.0 17.5 1.7 20.0 2.7

TABLE 7 Time/secs IC₅₀ K562 nM 0 580 5 35 10 40 15 20 20 18 25 17 30 1535 11 40 11 45 9 50 9 55 9 60 8

Example 10 Synthesis of Trans-CA-4 Bearing a Photocleavable Group

We next demonstrate the potential use of light to trigger the release ofCA-4 from a variety of non-toxic CA-4 derivatives. By way of example,two types of reaction were employed:

(1) Release of CA-4 from a trans-CA-4 derivative with E to Zisomerization.

(2) Release of CA-4 from a photolabile cis-CA-4 derivative.

As in some applications in the prior art, the insolubility of CA-4 inwater results in clinical difficulties, it would be advantageous toutilize a photocleavable group to impart water solubility to the system.

We first considered the fate of a trans-CA-4 derivative (2) bearing aphotocleavable water solubilising (PCWS) group (see the scheme below).When irradiated with ultraviolet light, two events can occur; (i) thePCWS group can be cleaved and (ii) trans to cis isomerisation can takeplace, (but perhaps not in that order). The overall effect of thesetransformations should be a dramatic increase in cytotoxicity.

It became apparent that whilst there are examples of the use ofphotocleavable groups in medical applications there are no examples oftheir use in this context. To demonstrate the validity of this approachwe used an ortho-nitrobenzyl derivative as a prototypical photocleavablegroup. A vanillin based compound, developed by Holmes et al. (C. Holmes,J. Org. Chem., 1997, 62, 2370.) as a solid phase linker, was used as atemplate for our photocleavable group. The synthetic strategy to thecoupling agent 7 is outlined in the following scheme.

The next step was to couple the benzyl bromide 7 to trans-CA-4. Thereaction gave the desired product E-8 in a 73% yield, as illustratedbelow. E-8 precipitated out of solution as the reaction to place and wassubsequently filtered and purified by recrystallisation. The couplingreaction was repeated using cis-CA-4 as the substrate—the reactionproceeded in a 69% yield.

As with the trans to cis photoinduced isomerisation study coupledtrans-CA-4 was dissolved in benzene and the resulting solution wasirradiated for a total of 20 minutes with aliquots removed at given timepoints to determine the isomeric ratios and the IC₅₀ value. The resultsobtained are detailed in Table 8.

TABLE 8 % coupled IC₅₀ K562 Time/mins trans-CA-4 (E-8) % trans-CA-4 %cis-CA-4 (nM) 0 100 — — >150000 0.5 93 7 — 14580 1.0 88 8 4 450 1.5 8012 8 90 20. 72 19 9 59 3.0 62 27 11 14 4.0 61 31 8 12 5.0 53 37 10 8 7.542 45 13 4 10.0 34 47 19 6 12.5 20 35 45 2 15.0 Unable to calculate* 217.5 Unable to calculate* 4 20.0 Unable to calculate* 3

The results show that the photocleavage of trans-CA-4 is the first eventto occur, followed soon after by the isomerisation of trans-CA-4 tocis-CA-4. This process is accompanied by an even more dramatic increasein the cytotoxicity than was seen previously, this is largely becauseE-8 is much less cytotoxic than trans-CA-4, in fact it is 5 times lesscytotoxic. This is highly beneficial if E-8 were to be used as a CA-4prodrug. These results also highlight the speed with which the processoccurs. After just 5 minutes exposure to ultra violet light the IC₅₀value has fallen to single nanomolar figures, with slightly more than50% of the coupled starting material remaining and some 10% cis-CA-4present.

Once again it was desirable to investigate the effect of the coupledtrans-CA-4, E-8 upon irradiation in the presence of cancer cells.Therefore, K562 cells were dosed with a known concentration of coupledtrans-CA-4 E-8 and subsequently exposed to ultra violet radiation forspecific lengths of time and the IC₅₀ value determined. As with theprevious in situ experiment a series of control experiments wereperformed simultaneously to verify that any positive results obtainedwere due to the effect of ultra violet light on the drug candidate andnot its effect on the cells. These consisted of i) exposing K562 cellsonly (i.e. cells which were not dosed with any potential drug candidate)to ultra violet light, ii) preventing K562 cells dosed with drugcandidate from being exposed to ultra violet light, and iii) preventingK562 cells only (i.e. cells which were not dosed with any potential drugcandidate) from being exposed to ultra violet light. In all threecontrol experiments no cytotoxic effect was observed. The results of themain experiment are illustrated in Table 9. It is apparent that the samegeneral trend, with respect to the IC₅₀ values is seen in both the exsitu and the in situ experiments.

TABLE 9 Time/Mins IC₅₀ K562 (nm) 0 >100000 0.5 1540 1 450 1.5 250 2 1303 40 4 11 5 9 7.5 5 10 2 12.5 3 15 3 17.5 2 20 2

We needed to demonstrate that the photo by-product, thenitrosobenzaldehyde, 10 did not possess any cytotoxicity and thereforewas not contributing to the results obtained for the photoactivation ofcoupled trans-CA-4. The synthesis of the nitroso compound, 10 wasachieved by exposing compound 9 to ultra violet light for 10 minutes andthe desired compound was isolated from the resulting reaction mixturevia column chromatography (see the scheme below). The compound 10 lackedsignificant cytotoxicity.

Having convincingly established the feasibility of prodrugphotoactivation, the final objective was to render the coupledtrans-CA-4 compound, E-8 water-soluble. A water soluble prodrug of CA-4would clearly have clinical potential. Therefore, a water-solublederivative was synthesised in an attempt to overcome this problem. Thewater-soluble derivative was prepared from the methyl ester, E-8 by asimple hydrolysis reaction using NaOH in ethanol. However, due topurification problems the carboxylic acid, E-11 was isolated andsubsequently deprotonated with K^(t)OBu, the water-soluble salt wasisolated using ion-exchange chromatography.

The in situ study of the water-soluble derivative of trans-CA-4 wasperformed in the same manner as those described previously and theresults can be seen in Table 10. The results obtained suggest that thewater-soluble derivative, E-12 behaves in a similar fashion as themethyl ester derivative, E-8 when irradiated with ultra violet light inthe presence of cancer cells. The IC₅₀ values obtained are similar tothose obtained for the in situ study of the methyl ester derivative,E-8.

TABLE 10 Time/Mins IC₅₀ K562 (Nm) 0 >50000 0.5 1010 1 410 1.5 300 2 1603 50 4 58 5 46 7.5 22 10 16 12.5 12 15 10 17.5 9 20 8

Example 11 Further Biological Experiments

The effect of compounds 97-64H and 97-96 (quinone compound) was testedon H460 human lung xenograft grown subcutaneously by injecting equitoxicdoses of the compounds (0.75×MTD). Twenty four hours following injection(i.p.) both compounds caused extensive damage to the tumours which wasconsistent with necrosis caused by destruction of vasculature.

The results from testing compounds of the invention in cell based assaysare reported in Tables 12 to 27 below.

The references cited herein are incorporated by reference.

TABLE 11 P388 1C₅₀ A2780 1C₅₀ H460 1C₅₀ K562 Tub Ass 1C₅₀ Colchicine %HUVEC 1C₅₀ Compound (μM) (μM) (μM) (μM) (μM) Inhibition (μM)Permeability Comb A-4 (1) 0.0026 0.00072 1.51 2.4 12 0.001 7.1, 6.997-64H 0.000489 0.00366 <1.25, 12.5 79, 70 .0024 5.8 97-64L 0.1710.113 >100 43 NT 1.0 97-65 0.18 0.095 100 NT 0.35 3.4 96-188 0.32 .081NT NT 96-167 0.0008 0.00037 9 3.8 0.04 5.5 97-07 0.0023 0.00147 2.78 10 6.6, 12.3 97-13H 0.0326 0.0179 .032-.035 >100 84.2 0.045 4.3, 3.2 98-21.000935 .00066 <1.25 1.4 0.0095 4.5 97-96 0.57 0.19 .038 <1.25 82, 240.23, 0.22 2.2 98-23 2.18 2.15 4.37 NT NT 98-35H .00328 .00144 6.25 50.016 7.4 98-29 0.45 0.38 >100 53 0.43 3.9 99-03H 0.19 0.12 0.06 6000-82 .06 0.05 7.2 39.5 00-105 0.02 >100 3.6 17a 0.2 17b 6 19 0.04 1.820 7 1.8 23a 0.1 23b 0.79 27 9 28 0.021 1.5 31a 0.018 7.5 32a 0.044 1845 0.12 1.8 NT = NOT TESTED; ‘H’ COMPOUNDS ARE CIS, ‘L’ COMPOUNDS ARETRANS. Stilbenes synthesised by either Wittig or 2-step synthesisroutes. Quinone synthesised as in Section 9.

TABLE 12 Biological activity of stilbenes with 3,4,5-trimethoxysubstitution on the A ring

Compound MTT (P388) MTT (K562) number R₂ R₁ Config IC₅₀ (μM) IC₅₀ (μM)MA IC₅₀ (μM) CD IC₅₀ (μM) CA4 (15) OH OMe Z 0.003 0.001 0.175 3 32 H OMeZ ND 0.004 0.2 5.5 90 F OMe Z ND 0.01 0.085 2.8 93 Br OMe Z ND 0.001 0.410 96 E ND 7.83 >10 >25 101 OH H Z 0.3 0.14 >10 >25 102 E 34 22 >10 >25103 Me Me Z 0.1 0.04 2 >25 104 E 20 35.8 >10 >25

TABLE 13 Growth inhibition studies using HUVEC HUVEC Drug IC₅₀ (μM) CA4(15) 0.0026 90 0.004 32 0.006 93 0.067

TABLE 14 Biological activity of stilbenes with 3,4,5-trimethylsubstitution on the A ring

Compound MTT (K562) MA CD number R IC₅₀ (μM) IC₅₀ (μM) IC₅₀ (μM)Combretastatin A-4 (15) 0.001 0.175 3 117 H 0.31 0.650 >25 120 F 0.140.700 10 133 OH 0.020 0.120 10

TABLE 15 Growth inhibition studies using HUVECs HUVEC Drug IC₅₀ (μM)117 >1 120 0.46 133 0.05

TABLE 16 K562 cell cycle analysis % of cells with DNA content = 2nor >2n % of cells with % of cells % of cells % of cells Drug DNA content<2n in G₀-G₁ in S phase in G₂-M 117 15 3 10 87 120 23 6 17 77 133 6 3 691

TABLE 17 Biological activity of stilbenes with 3,4-dimethyl substitutionon the A-ring

Compound MTT (P388) MTT (K562) number R R¹ Config IC₅₀ (μM) IC₅₀ (μM) MAIC₅₀ (μM) CD IC₅₀ (μM) Combretastatin A-4 (15) Z 0.003 0.001 0.175 3 137H Me Z 2 3.4 >10 >25 138 E 17 >50 >10 >25 139 NO₂ OMe Z 20 1.8 >10 >25140 E >50 24 >10 >25 141 H OMe Z 4.9 1.6 >10 >25 142 E 20.5 >50 >10 >25143 Br OMe Z 2.5 1.15 >10 >25 144 E >50 >50 >10 >25 146 OH OMe Z 0.40.07 3.09 12.5 147 F OMe Z 1.7 0.09 6.57 >25 148 E 36 2.5 >10 >25 149 FMe Z 7.2 0.39 >10 >25 150 E 40 >50 >10 >25 151 NO₂ Me Z 9.7 1.5 >10 >25152 E >50 >50 >10 >25

TABLE 18 K562 cell cycle analysis % of cells % of cells with DNA content= 2n or >2n with DNA % of cells % of cells % of cells Drug content <2nin G₀-G₁ in S phase in G₂-M Combretastatin 7 3 6 92 A-4 (15) 146 31 8 2765 147 28 10 33 57

TABLE 19 Biological activity of stilbenes with 3,4,5-triethoxysubstitution on the A-ring

Compound Con- MTT (K562) MA CD number R fig IC₅₀ (μM) IC₅₀ (μM) IC₅₀(μM) Combretastatin A-4 (15) Z 0.001 0.175 3 247 Br Z 0.6 >10 >25 248E >50 >10 >25 249 H Z 0.5 >10 >25 250 E >50 >10 >25 251 F Z 0.0441.25 >25 252 E >50 >10 >25 253 OH Z 0.018 0.50 15.5 254 E 0.2 >10 >25255 Br Z 0.6 >10 >25 256 E >50 >10 >25 257 Cl Z 0.45 >10 >25 258 E 7 >10>25

TABLE 20 Growth inhibition studies using HUVECs HUVEC drug IC₅₀ (μM) 2530.05 251 0.19

TABLE 21 K562 cell cycle analysis % of cells with DNA content = 2nor >2n % of cells with % of cells % of cells % of cells Drug DNA content<2n in G₀-G₁ in S phase in G₂-M 253 4 3 6 92 251 24 7 18 75

TABLE 22 Biological activity of stilbenes with substitution on theolefinic bond

Com- MTT MA CD pound (K562) IC₅₀ IC₅₀ number R¹ R² Config IC₅₀ (μM) (μM)(μM) Combretastatin A-4 (15) Z 0.001 0.175 3 208 Me OTBDMS Z 0.2 1.5 >25209 E 6 >10 >25 210 Me OH Z 0.04 0.13 6 211 E 0.7 >10 >25 213 Me H Z 0.11.3 >25 214 E 0.8 >10 >25 217 Et OTBDMS Z 0.5 >10 >25 218 E 3.4 >10 >25219 Et OH Z 0.12 0.13 >25 220 E 4 >10 >25  80 CO₂H OH E >50 >10 >25  81CO₂Me OH E >50 >10 >25  82 CH₂OH OH E >50 >10 >25

TABLE 23 Growth inhibition studies using HUVECs HUVEC Drug IC₅₀ (μM) 2100.09 213 0.35 219 0.22

TABLE 24 K562 cell cycle analysis on double bond substituted analogues %of cells with DNA content = 2n or >2n % of cells with % of cells % ofcells % of cells Drug DNA content <2n in G₀-G₁ in S phase in G₂-M 210 235 22 73 213 23 5 21 73 219 24 7 19 74

TABLE 25 Biological activity of stilbenes with substitution on thedouble bond

Compound MTT (P388) MTT (K562) number Config R R¹ IC₅₀ (μM) IC₅₀ (μM) MAIC₅₀ (μM) CD IC₅₀ (μM) 200 E H Me 7 2 9 >25 210 Z Me H ND 0.04 0.13 6211 E Me H ND 0.7 >10 >25

TABLE 26 Biological activity of monofluoro prodrug precursors

Com- MA CD pound Con- MTT (K562) IC₅₀ IC₅₀ number R R¹ fig IC₅₀ (μM)(μM) (μM) Combretastatin A-4 (15) Z  0.001 0.175 3 240 OTBDMS F Z0.5 >10 >25 241 E 10   >10 >25 242 OH F Z  0.02 1.25 9 243 E 5   >10 >25 90 OMe F Z  0.01 0.085 2.8  18 OH OH Z   0.04^(a) 4-5 22 ^(a)L1210murine leukaemia cell line

TABLE 27 K562 cell cycle analysis % of cells with DNA content = 2nor >2n % of cells with % of cells % of cells % of cells Drug DNA content<2n in G₀-G₁ in S phase in G₂-M 242 29 7 22 71 90 16 5 16 79

What is claimed is:
 1. A compound represented by the structural formula:

wherein: the zigzag line indicates that the compound can be cis ortrans; X is selected from hydroxyl, nitro, aryl, heteroaryl, alkyl,alkoxy, CHO, COR, halogen, haloalkyl, NH₂, NHR, NRR′, SR, CONH₂, CONHR,CONHRR′, O-aryl, O-heteroaryl or O-ester; R₁ is selected from alkyl,CHO, alkoxy, NH₂, NHR, NRR′, SR, CF₃ or halogen; R₂ and R₃ areindependently selected from hydrogen, alkyl, alkoxy, hydroxyl, NH₂, NHR,NRR′, SR, haloalkyl or halogen; R₄ and R₅ are independently selectedfrom hydrogen, alkyl, CH₂NHCOR″ or CH₂CONHR″; and, R₆, R₇ and R₈ areeither all alkyl or all alkoxy; wherein at least one of the substituentsR₄ and R₅ is an alkyl group; and wherein R and R′ are independentlyselected from C₁₋₁₀ alkyl groups and R″ is a C₁₋₁₀ alkyl group, arylgroup or heteroaryl group; or a salt, an ester, a free acid or base or ahydrate thereof.
 2. The compound of claim 1 which is the cis orZ-isomer.
 3. The compound of claim 1 which is the trans or E-isomer. 4.The compound of claim 1, wherein the alkyl group R₄ and/or R₅ is amethyl or ethyl group.
 5. The compound of claim 1, wherein the compoundis selected from one of the compounds having said structural formulawherein (i) R₁ represents methoxy, R₂, R₃ and R₄ each representhydrogen, R₅ represents methyl, R₆, R₇ and R₈ each represent methoxy andX represents hydroxy; or (ii) R₁ represents methoxy, R₂, R₃ and R₄ eachrepresent hydrogen, R₅ represents ethyl, R₆, R₇ and R₈ each representmethoxy and X represents hydroxy.
 6. A compound represented by thestructural formula:

wherein: the zigzag line indicates that the compound can be cis ortrans; X is selected from hydroxyl, nitro, aryl, heteroaryl, alkyl,alkoxy, CHO, COR, halogen, haloalkyl, NH₂, NHR, NRR′, SR, CONH₂, CONHR,CONHRR′, O-aryl, O-heteroaryl or O-ester; R₁ is selected from alkyl,CHO, alkoxy, NH₂, NHR, NRR′, SR or CF₃; R₂ and R₃ are independentlyselected from hydrogen, alkyl, alkoxy, hydroxyl, NH₂, NHR, NRR′, SR,haloalkyl or halogen; R₄ and R₅ are independently selected fromhydrogen, alkyl, CH₂NHCOR″ or CH₂CONHR″; and, R₆, R₇ and R₈ are allalkyl groups; and wherein R and R′ are independently selected from C₁₋₁₀alkyl groups and R″ is a C₁₋₁₀ alkyl group, aryl group or heteroarylgroup; or a salt, an ester, a free acid or base or a hydrate thereof. 7.The compound of claim 6, wherein the R₆, R₇ and R₈ groups are methyl,ethyl or propyl groups.
 8. The compound of claim 6, wherein the compoundis selected from one of compounds having said formula wherein (i) R₁represents methoxy, R₂, R₃, R₄ and R₅ each represent hydrogen, R₆, R₇and R₈ each represent methyl and X represents fluorine; or (ii) R₁represents methoxy, R₂, R₃, R₄ and R₅ each represent hydrogen, R₆, R₇and R₈ each represent methyl and X represents hydroxy.
 9. A compoundrepresented by the structural formula:

wherein: the zigzag line indicates that the compound can be cis ortrans; R₁ is selected from alkyl, alkoxy, NH₂, NHR, NRR′, SR, CF₃, CHOor halogen; R₂ and R₃ are independently selected from hydrogen, alkyl,alkoxy, hydroxyl, NH₂, NHR, NRR′, SR, haloalkyl or halogen; R₄ and R₅are independently selected from hydrogen, alkyl, CH₂NHCOR″ or CH₂CONHR″;and, R₆, R₇ and R₈ are independently selected from hydrogen, alkyl oralkoxy; and, wherein R and R′ are independently selected fromsubstituted or unsubstituted C₁₋₁₀ alkyl groups and R″ is a substitutedor unsubstituted C₁₋₁₀ alkyl group, aryl group or heteroaryl group; or asalt thereof; wherein X is a group represented by:

wherein BOC represents a t-butoxycarbonyl group and the A group is anaturally occurring amino acid side chain.
 10. The compound of claim 9,wherein the BOC amino acid ester comprises Phe, Ile, Gly, Trp, Met, Leu,Ala, His, Pro, D-Met, D-Trp, or Tyr.
 11. The compound of claim 9,wherein the amino acid is Phe, the A group is —CH₂Ph.
 12. A compositioncomprising the compound of claim 1 and a pharmaceutically acceptablecarrier.
 13. A composition comprising the compound of claim 6 and apharmaceutically acceptable carrier.
 14. A composition comprising thecompound of claim 9 and a pharmaceutically acceptable carrier.
 15. Amethod for the treatment of cancer or a condition characterized byabnormal proliferation of the vasculature in a patient in need of saidtreatment by administering a therapeutically effective amount of acompound of claim
 1. 16. The method of claim 15, wherein said compoundis administered for treatment of a condition characterized by abnormalproliferation of the vasculature that is selected from the group ofdiabetic retinopathy, psoriasis and endometriosis.
 17. A method fortreatment of cancer or a condition characterized by abnormalproliferation of the vasculature in a patient in need of said treatmentby administering a therapeutically effective amount of a compound ofclaim
 6. 18. The method of claim 17, wherein said compound isadministered for treatment of a condition characterized by abnormalproliferation of the vasculature that is selected from the group ofdiabetic retinopathy, psoriasis and endometriosis.
 19. A method for thetreatment of cancer or a condition characterized by abnormalproliferation of the vasculature in a patient in need of said treatmentby administering a therapeutically effective amount of a compound ofclaim
 9. 20. The method of claim 19, wherein said compound isadministered for treatment of a condition characterized by abnormalproliferation of the vasculature that is selected from the group ofdiabetic retinopathy, psoriasis and endometriosis.
 21. The compound ofclaim 1, having the formula: